• Antisense oligodeoxynucleotide inhibition of sodium-calcium exchange in cardiac and arterial myocytes

      Slodzinski, Martin Konrad; Blaustein, Mordecai P. (1998)
      The cardiac NCX protein half-life and the effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the cardiac and arterial Na - Ca2+ exchanger (NCX) were studied in rat myocytes. Low concentrations (0.5 muM) of chimeric, phosphorothioated AS-oligos targeted to the NCX1 transcript were used to knock down NCX protein and activity. Control cardiac myocytes incubated without oligos or with scrambled (NS-) oligos exhibited spontaneous Ca2+ transients (measured with fura-2). The cytosolic free Ca2+ concentration ([Ca2+]i) rose in control cells when external Na (Nao) was removed. In contrast, 60% of cells treated with AS-oligos did not exhibit spontaneous Ca2+ transients or respond to Na-free medium. In all cells, 50 mM K increased [Ca2+]i. NCX protein was reduced by 50% in cells treated with AS-oligos for 7 days, but was not reduced after only 2 days. The biochemical and protein half-life are consistent with the physiological evidence of about 60% knock down of cardiac NCX activity after 4 days. Arterial myocytes proliferated normally in the presence of NS-, AS-, or no oligos, without detectable quantitative or morphological differences. Reduction of Nao to 5 mM, which promotes the Nai-dependent component of Ca2+ influx caused [Ca2+]i to rise in controls and in NS-oligo treated cells, but not in AS-oligos treated cells. Incubation with 1 mM ouabain to raise Nai augmented Nai-dependent component of Ca2+ influx in controls and NS-oligo treated cells, but the AS-oligo treated cells still did not respond with a rise in [Ca2+]i. Nevertheless, all three groups of cells exhibited increases in [Ca2+]i in response to 10 muM serotonin (5-HT). With La3+ inhibition of the PMCA pump and Na-free media (to inhibit NCX), CAF and CPA elevated [Ca2+]i; the Nao-dependent component of Ca2+ efflux was selectively inhibited by AS-oligos, but not in sense (S-), or NS-oligos treated cells or controls. The knock down of NCX activity in arterial myocytes was reversible. The La3+-sensitive (PMCA pump) component of Ca2+ efflux was unaffected in AS-oligos treated cells. The transient [Ca2+]i elevations by 5-HT (10 muM, 30 s) at low frequency (15 min) in AS-oligo treated cells were no different from those in controls and NS-oligos treated cells in terms of [Ca2+]i amplitude, and rates of rise and recovery. [Ca2+]i recovery in AS-oligo treated cells was markedly slowed when the PMCA pump was inhibited by La3+. When cells were stimulated at higher frequency (3 min), however, the peak amplitudes of the 2nd and 3rd responses were larger, and [Ca2+]cyt returned to baseline more slowly in AS-oligo treated cells than controls. Peak 5-HT evoked responses in controls, but not AS-oligo treated cells, were augmented >2-fold in Na-free media. The response to 5-HT was tested immediately after removal of external Ca2+ before and after 10 min incubation with 1 muM ouabain. Ouabain significantly augmented the 5-HT induced rise in [Ca2+]cyt in controls, but had no effect on AS-oligos treated cells. The latter observation implies that the arterial myocytes NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness, while the results of the repetitive stimulation experiments suggest that the NCX may be important during tonic activation.
    • Application of radiotelemetry in pharmacokinetic-pharmacodynamic modeling of procainamide and N-acetylprocainamide in sedentary and exercise trained rats

      Kharidia, Jahnavi Sanjay; Eddington, Natalie D. (1996)
      Understanding the pharmacokinetics (PK) and pharmacodynamics (PD) of a new drug in an appropriate animal model provides a scientific framework for efficient and rational drug development. Cardiovascular measurements from small animals are difficult or impossible to obtain. The conventional methods, chemical restraint, physical restraint, cuff blood pressure and tethering used to obtain cardiovascular PD have limitations. These methods affect physiological function of animals and introduce stress artifact into data. Recently, radiotelemetry has been proposed as a new method to obtain cardiovascular PD from small animals. The objective of this research was to investigate the application of radiotelemetry in the pharmacokinetic/pharmacodynamic (PK/PD) modeling of cardiovascular agents in small animals. PD of various cardioactive agents such as procainamide (PA) and its active metabolite, N-acetylaprocainamide (NAPA) were evaluated. Additionally, one of the objectives of this dissertation was to evaluate the effects of exercise training on the pharmacokinetics of a model substrate of N-acetylation reactions (phase II enzymes). PA was chosen as a model drug for these studies. Radiotelemetry avoided the stress associated with animal handling and the use of anesthesia during PD studies, which allowed for the attainment of higher quality and more accurate data. PA displayed a significant increase in QT, QR and QS intervals from the baseline. The QT prolongation has been used as a surrogate marker in PA clinical studies. The PK of PA and NAPA were best described by a one compartment model. An Emax model was able to accurately describe the relationship between PA concentration and the QT interval. Effects of exercise training on the PK as well as PD of PA and NAPA were evaluated using radiotelemetry. Rats were exercised for four weeks by treadmill running. These studies noted that four weeks of exercise training did not alter the PK or PD of PA and NAPA suggesting that exercise training may not have significantly affected N-acetyltransferase. In conclusion, radiotelemetry allows the prediction of the PK/PD relationship observed in clinical studies by conducting such studies in small animals.
    • Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle

      Cheng, Heping (1995)
      Spontaneous local increases in the concentration of intracellular calcium ( (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar}), called "calcium sparks", were detected in quiescent rat heart cells with a confocal laser scanning microscope and the fluorescent calcium indicator fluo-3. A calcium spark is associated with an elevation of (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar} by {dollar}\sim{dollar}200 nM within a volume of {dollar}\sim{dollar}10 fl, and to decline with an half time of {dollar}\sim{dollar}20 msec. Estimates of calcium flux associated with the local increase in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar} suggest that calcium sparks arise from the spontaneous openings of single or a few sarcoplasmic reticulum (SR) calcium-release channels (also known as ryanodine receptors, RyRs) acting in concert, a finding supported by ryanodine modification of spark kinetics. Thus calcium sparks represent the functional elementary events of the SR release of calcium. By measuring the occurrence of calcium, the in vivo open probability of RyR/channels is shown to be around 0.0001 S{dollar}\sp{lcub}-1{rcub}{dollar} at resting (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar}. It is generally agreed that during cardiac excitation-contraction (EC) coupling, calcium release from the SR is triggered by the sarcolemmal calcium current (I{dollar}\rm\sb{lcub}Ca{rcub}{dollar}) via the calcium-induced calcium release mechanism. However, it is unclear how a mechanism with intrinsic positive feedback can provide graded responses. This work reveals, for the first time, that at the microscopic level, EC coupling takes the form of I{dollar}\rm\sb{lcub}Ca{rcub}{dollar}-evoked calcium sparks. Direct visualization of evoked calcium sparks was possible when I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} was reduced by calcium channel antagonists D600 or cadmium, or during small ramp depolarization under whole-cell voltage-clamp conditions. These evoked calcium sparks resemble spontaneous calcium sparks observed at rest, in amplitude and in spatio-temporal properties. The activation of calcium sparks is controlled by local I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} in a stochastic manner. Once activated, calcium release from the SR during a calcium spark is essentially independent of the triggering calcium influx and does not activate neighboring SR release sites. These novel findings are used to develop a new mechanistic model for cardiac EC coupling in which the graded amplification of the triggering I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} by the SR can be explained by altering the extent of spatial and temporal summation of the elementary release events.
    • Calcium transport in intact mammalian fast-twitch and slow-twitch skeletal muscle fibers

      Carroll, Stefanie Leigh; Schneider, Martin F. (1996)
      Calcium ions that are released from the Sarcoplasmic Reticulum (SR) upon electrical stimulation bind to troponin C of the thin filament structure, and initiate contraction of the skeletal muscle fiber. In order for the muscle fiber to relax, calcium must dissociate from troponin C and be removed from the cytoplasm by reuptake via the SR Calcium ATPase or by binding to cytoplasmic proteins. The intricacies of the calcium removal system in intact mammalian fibers have not been elucidated. The goal of my thesis project was to characterize the calcium removal system in mammalian intact skeletal muscle fibers, and determine the contribution of the individual components involved, such as the SR calcium ATPase, troponin C, and parvalbumin. Rat fast-twitch flexor digitorum brevis fibers (FDB) and slow-twitch soleus fibers are enzymatically dissociated and suspended in low melting temperature agarose gel to minimize fiber movement during fluorescence recordings. FDB fibers and soleus fibers are loaded with fura-2 (cell permeant form) and electrically stimulated by 1 to 40 pulses. Florescence signals are recorded at 380 (calcium sensitive) and 358 (calcium insensitive) nm excitation. Ca2+ is calculated assuming non-instantaneous equilibrium with fura-2. The rate constant of calcium decay decreased significantly with increasing stimulation duration in the FDB fibers, but remained relatively constant in the soleus fibers. This is due to expected differences in parvalbumin concentration between fast-twitch and slow-twitch fibers. In fast-twitch fibers parvalbumin becomes increasing saturated by calcium with increasing stimulation durations and can no longer contribute to the decay of calcium. However, there is negligible amounts of parvalbumin in slow-twitch fibers, therefore they do not exhibit this slowing of calcium decay effect. Quantification of the SR calcium ATPase, troponin C and parvalbumin content, using SDS page and immunoblotting techniques confirmed that there was a significant difference in the concentration of parvalbumin between rat FDB (1.2 mM calcium binding site concentration) and soleus fibers ({dollar}<{dollar}50 {dollar}\mu{dollar}M calcium binding site concentration). Unexpectedly there was no significant difference in the concentration of SR calcium ATPase, and troponin C. The values determined by the gel and immunoblot Quantification were well supported by preliminary modeling analysis of the Ca2+ decay. In conclusion, there are significant differences in the decay of Ca2+ in rat fast-twitch and slow-twitch muscle, which is due to differences in parvalbumin concentration. This indicates that parvalbumin has a significant role in the decay of calcium in mammalian skeletal muscle fibers.
    • Calcium(2+)-signaling in cardiac muscle: From development to heart failure

      Dilly, Keith Wayne; Lederer, W. Jonathan (2001)
      Some changes observed in heart failure include altered ultrastructure, defects in excitation contraction coupling (ECC) and serious changes in beta-adrenergic signaling. I have examined these topics with an emphasis on cellular Ca 2+ signaling. Using immunohistochemical techniques combined with fluorescence confocal laser scanning microscopy (FCLSM) I have examined the localization and distribution of structural components of ECC in isolated ventricular myocytes from neonatal rats, and rat models of hypertrophy (Dahl SS/jr) and heart failure (SHHF). Using whole cell patch clamp techniques to record membrane voltage or currents, and simultaneously measuring intracellular [Ca2+] i using fluorescent Ca2+ sensitive indicators and FCLSM I have examined cardiac function in MLP-/- and transgenic V12HRas murine models of heart failure. I also examined the effectiveness of therapeutic strategies at preventing ECC defects in these models, Even at the early stages of development examined, primitive striations of a number of ECC protein structures were observed. Striations of sarcolemmal membrane (T-tubules) appear to form from the outside inwards, whereas proteins localized more intracellularly are first seen more internally. Structural changes observed in hypertrophic and failing myocytes include altered distribution of the Na 2+/Ca2+ exchanger. Increased Na2+/Ca 2+ exchanger density at the external surface is seen in these myocytes. In Ras myocytes unchanged Ca2+ currents (ICa(L)), decreased [Ca2+]i transients, defective ECC and action potential (AP) alterations were seen. Crossing Ras and PLBKO mice prevented AP alterations. In MLP-/- unchanged ICa(L ) and Ca2+ spark characteristics but decreased [Ca 2+]i transients, contractile responses and defective ECC were seen. Cellular defects were prevented in MLP-/- mice expressing a cardiac-targeted transgene blocking the function of beta-adrenergic receptor kinase-1 (betaARKct). These data suggest both defective SR function and down-regulation/de-sensitization of beta-adrenergic receptors play a pivotal role pathogenesis of heart failure. As such, PLBKO and betaARKct may prove effective therapies for preventing and possibly rescuing the cellular defects seen in heart failure. The mechanism(s) responsible for targeting ECC proteins to specific intracellular localizations remains to be discovered. Further studies on these and other methods for preventing and reversing Ca 2+ signaling defects seen in heart failure may provide valuable therapeutic tools for human heart failure treatment.
    • Characteristics and regulation of aspartate transport systems in rat ventral prostate epithelial cells

      Lao, Lixing; Franklin, Renty B.; Costello, Leslie (1992)
      A unique characteristic and function of rat ventral prostate, like human prostate, is the accumulation and secretion of high levels of citric acid. Aspartate is a proposed four-carbon precursor of citrate via transamination. Replenishment of endogenous aspartate requires continuous uptake of aspartate from circulation. This study was designed to identify aspartate transporters in isolated rat ventral prostate epithelial cells. The results indicated that two aspartate transporters, a high affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.01 mM) and a low affinity transporter (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.8 mM) exist in these cells. Both transporters are Na{dollar}\sp+{dollar}-dependent and pH sensitive. The optimal pH for the high affinity transporter is about 7.5, whereas for the low affinity transporter the optimum is between 6.5 and 7.0. The high affinity transporter is also temperature dependent. Competitive inhibitory studies indicate that L-aspartate uptake by the high affinity transporter is inhibited by L-glutamate and D-aspartate, but not by L-alanine and L-lysine. The low affinity system is inhibited by D-aspartate, but not by L-glutamate or L-alanine. These different characteristics suggest that the high affinity and the low affinity transporters are two distinct systems. The high affinity aspartate transporter is sensitive to the Na{dollar}\sp+{dollar}-K{dollar}\sp+{dollar} ATPase inhibitor vanadate but less sensitive to ouabain. This suggests that an ouabain-insensitive Na{dollar}\sp+{dollar}-ATPase exists on the cell membrane. High affinity aspartate uptake is not dependent on K{dollar}\sp+{dollar}. However, a Na{dollar}\sp+{dollar}-H{dollar}\sp+{dollar} antiport might be involved. Aspartate uptake is stimulated by testosterone in vivo and in vitro. The in vitro effect is rapid and is inhibited by cycloheximide and actinomycin D. Prolactin also stimulated aspartate uptake independent of testosterone and is inhibited by cycloheximide. The high affinity aspartate transporter is subject to transstimulation by aspartate and citrate.
    • Characterization of angiotensin II and protein kinase C signalling pathways that regulate intracellular pH in neonatal rat ventricular myocytes

      Kohout, Trudy Ana; Rogers, Terry Birkby (1995)
      Angiotensin II (AngII) exerts many functional effects on the heart through the activation of protein kinase C (PKC) to affect contractility, and growth. It is now known that PKC is a family of 11 isoforms designated {dollar}\alpha{dollar}, {dollar}\beta{dollar}I, {dollar}\beta{dollar}II, {dollar}\gamma{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\xi{dollar}, {dollar}\eta{dollar}, {dollar}\theta{dollar}, {dollar}\lambda{dollar}, and {dollar}\mu{dollar}. To examine the effects of PKC on the heart, it was first necessary to characterize which isoforms are expressed in this tissue. A RT-PCR approach was developed to identify isoforms that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Cardiac cDNA was RT-PCR amplified and the products analyzed by a combination of restriction mapping and DNA sequencing which revealed the presence of only the {dollar}\alpha{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\eta{dollar}, and {dollar}\xi{dollar} isoforms cardiac myocytes. Since many cardioactive hormones modulate intracellular pH (pH{dollar}\sb{lcub}\rm i{rcub}{dollar}), the goal of this study was to determine if AngII and PKC altered pH{dollar}\sb{lcub}\rm i{rcub}{dollar} in cultured neonatal rat ventricular myocytes. pH{dollar}\sb{lcub}\rm i{rcub}{dollar} was monitored in single cells loaded with the fluorescent indicator c-SNARF-1 or BCECF. Superfusion with 100 nM TPA, a direct activator of PKC, induces an alkalinization of 0.06 {dollar}\pm{dollar} 0.01 pH unit and increased the initial rate of recovery from an imposed acid load by 2.20 {dollar}\pm{dollar} 0.36 fold. The alkalinization and transporter activation are HCO{dollar}\sb3\sp-{dollar}-independent and amiloride-sensitive indicating the involvement of the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} exchanger. Furthermore, Cl{dollar}\sp-{dollar} removal experiments revealed a TPA-stimulated 1.31 {dollar}\pm{dollar} 0.11 fold enhancement of the acid-loading HCO{dollar}\sb3\sp-{dollar}-/Cl{dollar}\sp-{dollar} exchanger. The increase in the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} activity compared to that of the HCO{dollar}\sb3\sp-{dollar}/Cl{dollar}\sp-{dollar} exchanger is consistent with the alkalinization observed. Stimulation of the myocytes with 100 nM AngII resulted in a rapid HCO{dollar}\sb3\sp-{dollar}-dependent, amiloride-insensitive alkalinization of 0.08 {dollar}\pm{dollar} 0.02 pH unit. AngII also increased the rate of acid extrusion by 3.67 {dollar}\pm{dollar} 0.50 fold in a HCO{dollar}\sb3\sp-{dollar}-dependent and Cl{dollar}\sp-{dollar}-independent manner, indicating the activation of the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar}-symport. The AngII activation of the symport is mediated through an AT{dollar}\sb2{dollar}-like signalling pathway since the pH{dollar}\sb{lcub}\rm i{rcub}{dollar} response was blocked by the AT{dollar}\sb2{dollar} receptor antagonist, CGP 42112A, and was unaffected by the AT{dollar}\sb1{dollar} inactivator, DTT. Superfusion of the myocytes with 5 {dollar}\mu{dollar}M arachidonic acid (ARA) mimicked the AngII-mediated alkalinization, suggesting further that ARA may mediate the response. Moreover, the AngII- and the ARA-induced responses were blocked with staurosporine, a PKC inhibitor. In summary, AngII activates the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar} symport through the AT{dollar}\sb2{dollar} pathway via ARA and possibly through PKC. Although TPA and AngII both alkalinize the cell, they do so through two distinct pathways, perhaps by activating different PKC isoforms.
    • Chronic pain and thalamic abnormalities after traumatic brain injury

      Parihar, Abhishruti; Keller, Asaf (2014)
      Traumatic brain injury (TBI) is an important public health issue in both military and civilian life. Many suffer from cognitive and motor deficits, as well as from excruciating, unrelenting chronic pain (TBI-Pain). TBI-Pain is associated with hypersensitivity to mild tactile and thermal stimulation of the face and scalp, a result of central sensitization, a process by which brain structures undergo maladaptive plasticity, resulting in abnormal activity of brain neurons. Recently central sensitization of the posterior thalamus (PO) has been implicated in chronic pain disorders, spinal cord injury and migraine. We therefore hypothesized that chronic pain after TBI is also associated with abnormal activity of the PO thalamus. Here, we tested this hypothesis using a novel model of blast-TBI with two unique features: (i) blast-TBI was performed in awake, unanesthetized rats, to simulate the human experience and to preclude anesthesia-induced dampening of post-injury increases in excitatory activity that is crucial for the development of central pain; (ii) only the cranium, rather than the entire body, was exposed to a collimated blast wave, with the blast wave striking the posterior cranium in the region of the occipital crest and foramen magnum. Testing for thermal hyperalgesia of the face (distal from direct injury) revealed that blast-TBI rats had a significantly lower tolerance to pain, compared to the control group. Consistent with the behavioral data, single unit electrophysiological recordings from PO showed an increase in the spontaneous and evoked firing rate of neurons from blast-TBI rats, compared to sham. These data support the hypothesis that blast-TBI is associated with hyperalgesia and maladaptive plasticity in the PO thalamus.
    • Chronic Sensory and Affective Craniofacial Pain After Blast-Induced Traumatic Brain Injury and Peripheral Nerve Injury in Rats

      Studlack, Paige Elizabeth; Keller, Asaf; Simard, J. Marc; 0000-0002-3626-1865 (2017)
      Thousands of military members suffer long-term consequences of blast-induced traumatic brain injury (Blast-TBI), including chronic head and face pain. Pain after blast-TBI usually manifests as post-traumatic headaches with a high degree of comorbid mood disorders, suggesting that the affective dimension of pain may burden survivors of blast-TBI. Here, we tested the hypothesis that an innovative model of the unique aspect of blast-TBI over blunt-force TBI, the primary blast injury, directed over the cranium sufficiently modeled long-term conditions of human blast exposure in rats. Rats exposed to cranium-directed primary blast-TBI demonstrated behavioral manifestations of ongoing pain, mechanical hyperalgesia, and cold allodynia three weeks after injury, recapitulating chronic facial pain in patients after blast-TBI. We predicted that maladaptive changes to pain-signaling and -processing nuclei in CNS would induce and maintain pain behavior after blast-TBI. We recorded single units in sensory pain-associated nuclei, the posterior nucleus of the thalamus (PO) and spinal trigeminal nucleus caudalis (SpVc), which have previously been causally associated with pain after spinal cord injury. We observed hyperexcitability at baseline of PO neurons after blast injury in absence of changes to evoked response to cutaneous noxious stimuli. Neuronal hyperexcitability in PO is not associated with persistent gliosis. Affective pain processing through the parabrachial complex (PB) occurs in parallel to information coding the sensory dimension of pain through PO. We assessed central changes to PB neuronal activity in a robust model of post-traumatic pain using the chronic constriction injury of the infraorbital nerve (CCI-ION). PB neurons, weeks to months after injury, are hyper-excitable in chronic pain, as shown by prolonged response after presentation of noxious cutaneous stimulation ("after-discharges"), previously observed to be causally-related to pain due to CCI-ION in SpVc. Further study of PB hyperexcitability in blast-TBI rodent models may elucidate the mechanism underlying blast-TBI-associated affective pain.
    • Effects of aging and fetal tissue transplantation on the patterns of expression of neuroendocrine and neurochemical outputs of the biological clock

      Cai, Aihua; Wise, Phyllis M. (1995)
      Circadian rhythmicity is a common physiological phenomenon. Many rhythms change with age. Since the suprachiasmatic nucleus (SCN) is the major neural circadian pacemaker, we hypothesized that changes in the circadian rhythms maybe due to changes in the SCN itself. In this dissertation, I asked two questions: (1) Does age affect the immediate early gene expression in the SCN? If so, can transplantation of fetal tissue containing the SCN restore normal function? (2) Does age change SCN-driven endocrine outputs, such as the hypothalamic-pituitary-adrenal cortex (HPA) axis; If so, can fetal SCN transplants restore normal function? The expression of immediate early genes (IEG), fos and jun, have been used as markers of neural activity in many studies. We used expression of IEG in the SCN as an index of neural activity of the SCN. Using immunocytochemistry, we detected a significant decrease in light-induced Fos and Jun-B expression in the SCN in middle-aged rats, and transplantation of fetal SCN tissue into middle-aged rats partially restored the IEG expression in hosts to that of young rats. We chose to study corticotropin releasing hormone (CRH) and proopiomelanocortin (POMC) gene expression in HPA axis. Using in situ hybridization histochemistry and solution hybridization-RNase protection assays, we detected that both diurnal rhythms of CRH mRNA in the paraventricular nucleus (PVN) and POMC mRNA in the anterior pituitary were abolished by the time animals were middle-aged. Transplantation of fetal SCN tissue into middle-aged rats restored a diurnal rhythm of CRH mRNA in the PVN, but the pattern was slightly altered. SCN transplants can restore a diurnal rhythm of POMC mRNA in the anterior pituitary, but the amplitude of the rhythm remained as low as that of middle-aged rats. In conclusion, aging affects rhythms of the SCN and SCN-driven endocrine outputs. These changes may not be permanent, since fetal SCN transplants can partially correct those changes.
    • Effects of experimental pulpitis on the expression of transient receptor potential channels on the rat trigeminal ganglia

      Duraes, Gabriela; Ro, Jin Y. (2010)
      Management of patients who present with tooth pain is one of the major challenges in dentistry. Further elucidation of mechanisms underlying tooth pain would lead to optimized management of these patients. Recent evidence suggests that Transient Receptor Potential (TRP) channels participate in pain sensation induced by chemical, thermal and mechanical stimuli. The aim of this study is to explore the molecular mechanisms underlying pulpitis by investigating the involvement of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels in tooth pain using an animal model. Experiments were performed on 42 male Sprague-Dawley rats. Pulpitis was induced by drilling the first maxillary molar of the animal and treating the cavity with either complete Freund's adjuvant (CFA) or saline. Naïve (untreated) rats were used as control. Trigeminal ganglia (TG) from both sides were extracted 1, 3 and 7 days after pulpitis induction and Western blot analysis was performed. The data was analyzed with one-way ANOVA or Kruskal-Wallis ANOVA on ranks depending on the outcome of normality test. TG from naïve and pulpitis-induced rats showed the expression of both TRPV1 and TRPA1. Increased expression of both receptors was observed in TG of rats treated with saline and CFA, compared to naïve (untreated) rats. However, results were not statistically significant due to a large variability. Potential factors that might have contributed to the variability, strengths and limitations of the pulpitis model employed in this study are discussed. Confirmation of our results with larger samples may provide a rationale for targeting these channels and reveal new therapeutic strategies for pulpitis.
    • Endocannabinoids and dopamine drive incentive learning processes underlying addiction vulnerability

      Bacharach, Sam; Calu, Donna J; 0000-0002-8036-7077 (2021)
      Phenotypic behavioral differences termed sign-tracking (ST) and goal-tracking (GT) differentially predict vulnerability to addiction and relapse. ST rats show enhanced cue sensitivity prior to drug experience that predicts greater discrete cue-driven drug-seeking compared to GT rats. Cue-evoked dopamine in the nucleus Accumbens (NAc) is a neurobiological signature of sign-, but not goal-tracking. Here, we examine an important regulator of the dopamine system; endocannabinoids, which primarily bind to the cannabinoid receptor-1 (CB1) in the central nervous system. We use pharmacological, optogenetic, and fiber photometry approaches to test the hypothesis that ventral tegmental area (VTA) CB1 receptor activation regulates extracellular concentrations of NAc dopamine to control sign-tracking. Rats are first trained in a Pavlovian lever autoshaping procedure to determine tracking group. We demonstrate that optogenetic inhibition of dopamine terminals in the NAc selectively reduce lever-directed behaviors, whereas intra-VTA CB1 receptor blockade both decreases lever-directed while increasing foodcup-directed behaviors. This change in response profile was coincident with increased dopamine levels in the NAc shell during the reward delivery period. These findings implicate VTA CB1 receptor signaling in maintaining Pavlovian cue-reward relationships in sign-tracking rats.
    • Evaluation of Multifunctional Compounds Possessing Anticholinergic and Antiglutamatergic Properties for Efficacy Against Soman Toxicity in Rats

      Schultz, Mark Kendall; Mong, Jessica Aurora (2015)
      Chemical warfare nerve agents (CWNA) such as soman (GD) are highly toxic organophosphorus cholinesterase inhibitors and pose a significant threat to both military and civilian populations. CWNA exposure causes seizure activity, which progresses to self-sustaining status epilepticus (SE) and becomes refractory to anticonvulsant treatment. SE is accompanied by neuropathology and functional behavioral impairments. Seizure activity is initiated through excessive stimulation of acetylcholine receptors and maintained by excessive glutamatergic stimulation, which causes pro-convulsive neuroplastic changes and excitotoxic damage. As these two neurotransmitter systems are crucial to the initiation and maintenance of CWNA-induced seizure activity, we hypothesized that multifunctional drugs with combined anticholinergic and antiglutamatergic activity could terminate seizure activity, and thereby prevent neuropathology and behavioral deficits. To test this hypothesis, we selected two drugs to be used as adjunct treatment in a rat GD-exposure model: caramiphen, an anticholinergic with antiglutamatergic activity, and ketamine, an antiglutamatergic with anticholinergic activity. Rats were exposed to 1.2 LD50 GD and treated with one of the test drugs in association with standard antidotal therapy consisting of atropine to block overactivation of muscarinic receptors, an oxime to reactivate GD-inhibited cholinesterase, and a benzodiazepine (midazolam or diazepam) to suppress acute convulsions. Subsequently, rats were subjected to a series of behavioral tests to assess vestibular-motor, locomotor, and sensorimotor function, as well as spatial learning and memory retention, fear memory, response inhibition, and interval timing behavior. Continuous EEG recordings were used to measure seizure activity. Brain tissue was assessed for neuropathological injury at the conclusion of each experiment. Results presented here demonstrate that the association of standard antidotal therapy with caramiphen within the first 10 min following seizure onset effectively attenuates the acute seizures, prevents the acute neurodegeneration, and mitigates most of the delayed neurological deficits that develop following the exposure to GD. They also reveal that administration of ketamine or a higher dose of caramiphen in combination with a benzodiazepine 30-40 min after onset of seizures counters the acute and most of the delayed neurotoxic effects of GD. In both cases, GD-exposed rats treated with caramiphen or ketamine in addition to the standard antidotal therapy had less neurological damage than those treated only with the standard therapy. It is noteworthy, however, that neither ketamine nor caramiphen fully attenuated the GD-induced impairments in vestibular-motor, locomotor, and sensorimotor tests. In sum, treatment of CWNA-induced seizure with a benzodiazepine and a multifunctional compound possessing both antiglutamatergic and anticholinergic properties is a viable strategy to terminate seizure, prevent neuropathology, and preserve many neurological functions.
    • Functional disorders in luteinizing hormone-releasing hormone and A1 noradrenergic neurons in androgen-sterilized rats

      Liaw, Jiin-Jia; Barraclough, Charles A. (1992)
      A single injection of androgen to 5-day old female rats renders these animals permanently sterile as adults. Previous studies suggest that the principle neuroendocrine malfunction in these androgen-sterilized rats (ASR) resides within the noradrenergic system and norepinephrine (NE) plays an important role in triggering preovulatory LHRH/LH surges. Although synaptic connections between NE and LHRH neurons are established prior to or at birth, it remains to be resolved whether these synapses are functional in ASR. In initial studies, we observed that the pituitary glands of ASR are less responsive to pulse injections of LHRH than normal rats. As well, responsiveness of LHRH neurons to electrochemical stimulation (ECS), icv infusion of norepinephrine (NE) or combined ECS + icv NE also are markedly diminished in ASR. When combined preoptic and A1 stimulations were performed, marked amplification of LH release occurred in both controls and ASR although less LH was released in ASR. Thus, LHRH-NE synapses are functional in ASR but the NE signal spontaneously is not presented to LHRH neurons. Consequently, spontaneous LH surges do not occur in ASR. LHRH neuronal responsiveness to NMDA stimulation also was found to be reduced in ASR. NMDA increased LHRH mRNA levels in the OVLT and rostral preoptic area in controls but not in ASR. These data suggest that a deficiency exists in the mechanisms responsible for transcription of LHRH mRNA in ASR. Other studies provided evidence that changes in levels of tyrosine hydroxylase (TH) mRNA in catecholamine neurons could serve as an index of increased activity within these cells. In proestrous rats, significant increases in TH mRNA levels were observed in A1 neurons during the afternoon hours when increases in hypothalamic NE turnovers occur. None of these events occurred in ASR. In summary, when the undifferentiated NE-LHRH-pituitary axis is exposed to high levels of androgen during a critical period in development, damage occurs at all levels within this axis to such an extent that spontaneous LH surges are permanently abolished and these animals become permanently sterile as adults.
    • Growth factor and cytokine regulation of the beta gene in rat astrocytes

      Hinkle, David Alan; Wise, Phyllis M. (1995)
      Basic fibroblast growth factor (FGF-2) and interleukin-l{dollar}\beta{dollar} (IL-1{dollar}\beta{dollar}) stimulate reactive gliosis and regulate neurotrophic factor expression in astrocytes. S100{dollar}\beta{dollar} is a putative neurotrophic factor which is over-expressed in reactive astrocytes. Therefore, we tested the hypothesis that FGF-2 and/or IL-1{dollar}\beta{dollar} would stimulate S100{dollar}\beta{dollar} gene expression. FGF-2 decreased S100{dollar}\beta{dollar} mRNA after 12 and 24 hours of treatment in cultured rat cortical astrocytes, but increased its levels after 7 days. IL-1{dollar}\beta{dollar} suppressed S100{dollar}\beta{dollar} mRNA levels after 24 and 48 hours, and continued to inhibit after 7 days of exposure. In combination, the effect of these two factors appeared synergistic. In C6 glioma cells, only FGF-2 suppressed gene expression. To assess indirectly whether alterations in transcriptional rate could explain the changes in mRNA, we measured levels of S100{dollar}\beta{dollar} primary transcript. FGF-2 decreased S100{dollar}\beta{dollar} nuclear primary transcript levels after 6 and 12 hours, but increased its levels after 48 hours. IL-1{dollar}\beta{dollar} decreased nuclear primary transcript after 48 hours. We further measured intracellular S100{dollar}\beta{dollar} protein levels to determine whether the alterations in mRNA were translated into parallel changes in the level of protein. FGF-2 did not suppress S100{dollar}\beta{dollar} protein levels after 1, 2, or 3 days of treatment, but increased it after 5 and 8 days. IL-1{dollar}\beta{dollar} and combination treatment did not significantly alter protein levels. Our results clearly demonstrate that FGF-2 and IL-1{dollar}\beta{dollar} influence the expression of the S100{dollar}\beta{dollar} gene, that this regulation appears to occur at the level of transcription, and that changes in mRNA are sometimes, but not always, reflected in changes at the level of protein. In a second study we determined whether the changes observed in vitro would also be seen in a lesion model for reactive gliosis in which both FGF-2 and IL-1{dollar}\beta{dollar} are elevated: the cortical contusion. Mild contusion bilaterally elevated S100{dollar}\beta{dollar} mRNA over sham levels in both the cortex and hippocampus of young adult, male rats. However, mRNA levels in sham animals decreased with time, making it unclear whether the contusions stimulated S100{dollar}\beta{dollar} or mitigated the inhibitory effect of sham. The contusion stimulated a robust elevation in GFAP mRNA, a "marker" of reactive gliosis, in both brain regions. Our data clearly demonstrate that contusion produces a vigorous glial response, and suggest that the mechanisms involved in the regulation of S100{dollar}\beta{dollar} and GFAP are different. This hypothesis is further supported by our in vitro finding that GFAP and S100{dollar}\beta{dollar} are differentially regulated in astrocytes by FGF-2 and IL-1{dollar}\beta.{dollar}* ftn*Originally published in DAI vol. 56, no. 9. Reprinted here with corrected author name.
    • Histogenesis of the neonatal rat ovary during follicle formation: Changing histo-architecture and collagen type IV gene expression

      Rajah, Roopmathy Roopa; Barrett, Charles P. (1994)
      In mammals, the hallmark of late ovarian histogenesis is follicle formation. However, fundamental issues regarding late histogenesis of the rat ovary have not been addressed. Accordingly, the objective of this research was to study and establish morphology, histology, and changing histo-architecture of the neonatal rat ovary during follicle formation. Light and electron microscopic observations and three dimensional reconstruction showed that: during follicle assembly, the oocytes in contact with each other became separated by intervening presumptive granulosa cells to form a single layer of cells around each oocyte, the first few follicles were formed during the second 24 hours after birth, follicle formation was nearly completed within the third 24 hours, there was a basal lamina wherever the epithelial cell apposed the mesenchyme, the basal lamina was not present wherever the presumptive granulosa cell apposed the oocyte, the shape of the neonatal rat ovary was similar to that of a cashew-nut, the intra ovarian rete tubules were in close proximity to the newly formed follicles, and there were dynamic changes in the histo-architecture of the neonatal rat ovary. Immunolocalization of collagen type IV, a marker for basal lamina, showed that: collagen type IV was present in the basal lamina of sex cords and follicles, the epithelial compartment changed from a cluster-like organization to a follicle-like organization, all the stages in follicle formation suggested by other researchers were found applicable to the neonatal rat ovary, and follicle formation began at the center of the ovary and later spread toward the periphery. In situ hybridization of mRNA coding for NC1 domain of {dollar}\alpha1{dollar}-chain of collagen type IV showed that: mesenchymal cells might be solely responsible for the synthesis of collagen type IV before and during follicle formation, presumptive granulosa cells might start synthesizing this protein only after they are assembled into a single layer of granulosa cells and bound by an enveloping basal lamina, and expression of this mRNA coding by granulosa cell might be a marker for commitment of the undifferentiated presumptive granulosa cell to the granulosa cell lineage.
    • Investigation of sigma and dextromethorphan-like neuroprotection using glutamate-induced LDH release, cellular morphology and dynamic calcium signaling

      Klette, Kevin Louis; Moreton, J. Edward (1995)
      The role of the putative sigma receptor in mediating neuroprotection against glutamate induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the sigma1, selective ligand (+)-3-PPP, all of the sigma receptor ligands tested were neuroprotective, preventing glutamate induced morphological changes and increases in LDH release. When corrected for relative sigma versus PCP binding site affinity, it appears that a positive correlation exists between neuroprotective potency and sigma1, site affinity. None of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC{dollar}\sb{lcub}50{rcub}{dollar} values. The effect of neuroprotective sigma ligands on the unique calcium responses evoked by glutamate, NMDA, potassium chloride (KCl) and trans-ACPD were investigated to elucidate the mechanism of sigma-mediated neuroprotection. In general, except for (+)-3-PPP all of the sigma ligands studied interfered with glutamate and NMDA induced (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} signaling, but, highly sigma{dollar}\sb1{dollar} selective ligands also lacking substantial PCP binding site affinity (i.e. carbetapentane, DTG and haloperidol) were much less effective in altering calcium influx induced by 80 {dollar}\mu{dollar}M glutamate. In contrast to glutamate, KCl (50 mM) produced changes in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} which were not neurotoxic to the neurons as measured by LDH release. Sigma ligands which lack substantial PCP site afflinity were very effective in altering KCl induced calcium signaling while the sigma/PCP site ligand (+)-cyclazocine was ineffective or, in the case of (+)-SKF 10047, much less effective. Similar to the effects of sigma ligands on KCl induced calcium dynamics, the sigma selective ligands DTG, haloperidol, (+)-pentazocine, and carbetapentane were very effective in altering intracellular calcium dynamics evoked by trans-ACPD while the sigma/PCP ligand (+)-SKF 10047 was ineffective or, in the case of (+)-cyclazocine, much less effective. Importantly, (+)-3-PPP, a non-neuroprotective sigma selective ligand, actually potentiated the calcium response elicited by trans-ACPD. The ability of sigma ligands applied at maximal neuroprotective concentrations to attenuate receptor and/or voltage-gated changes in calcium dynamics suggests that modulation of neurotoxic (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} plays a significant role in sigma-mediated neuroprotection. The unique modulatory effects of sigma ligands on the buffering of neuronal (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} will likely have numerous therapeutic applications in the treatment of CNS injury and other neurodegenerative disorders.
    • L-655, 708 Exerts Rapidly-Acting Antidepressant Efficacy in Rat Models of Depression

      Fischell, Jonathan Morris; Thompson, Scott M., Ph.D. (2014)
      The considerable morbidity and mortality associated with depression, combined with its extensive prevalence, makes optimally treating depression a key objective of mental health research. Currently, the first line of pharmacological treatment for depression are selective serotonin reuptake inhibitors (SSRIs). While SSRIs are effective tools in the treatment of depression, their value is substantially diminished by a delay of weeks to months before providing relief of the symptoms depression. Furthermore, SSRIs are only effective in one-half to two-thirds of patients. Investigation into alternative pharmacological treatments that could exert rapid antidepressant effects led to the discovery of ketamine, a non-competitive NMDA receptor antagonist. Unfortunately, ketamine is severely limited in its therapeutic value by its addictive and dissociative properties. Ketamine has been shown to increase excitatory neurotransmission in the hippocampus resulting in a long term enhancement of excitatory synaptic strength, an effect theorized to underlie its antidepressant efficacy. Another way to increase excitatory neurotransmission, is through the attenuation of GABAergic synaptic inhibition using compounds such as the α5 selective, GABAA receptor partial inverse agonist, L-655, 708. In this thesis, I have demonstrated that a single injection of L-655, 708 rapidly reversed a stress-induced impairment of sucrose preference and social interaction in a rat model of stress-induced depression. Associated with this reversal, L-655, 708 rapidly restored the strength of pathologically weakened AMPAR-mediated, but not NMDAR-mediated neurotransmission at temproammonic-CA1 synapses, measured electrophysiologically. Additionally, L-655, 708 rapidly increased S831 phosphorylation at these synapses. This finding indicated that an enhancement of AMPA receptor function may underlie the electrophysiological findings. Based on our previously proposed, excitatory synapse hypothesis of depression, we theorize that ability of L-655, 708 to restore excitatory synaptic strength underlies its efficacy as an antidepressant. Finally, L-655, 708 appears to be non-anxiogenic, as measured by open-field testing. Before now the antidepressant efficacy of compounds like L-655, 708 have never been investigated. We conclude that L-655, 708 may be a novel, effective, rapidly acting, and clinically viable treatment for depression.
    • Regulation of vascular endothelial growth factor gene expression by hypoxia

      Forsythe, Jo Ann; Koos, Robert D. (1996)
      Vascular endothelial growth factor (VEGF), a specific endothelial cell mitogen and regulator of microvascular permeability plays a central role in angiogenesis. Hypoxia stimulates VEGF gene expression in a variety of cultured cell types. The ovary is one of the few sites where angiogenesis normally occurs. Granulosa cells are probably exposed to hypoxic conditions during follicular development; this environment likely leads to enhanced VEGF secretion. Increased VEGF expression may play a role in the processes of ovulation and corpus luteum development. This dissertation investigates the hypoxic induction of VEGF expression in granulosa cells and characterizes the hypoxia response element in the VEGF gene.Using reverse transcription-polymerase chain reaction (RT-PCR), we have shown that rat ovarian granulosa cells exposed to hypoxia respond by increasing steady-state levels of VEGF mRNA. This increase is apparent within 3 h and is maximal by 12 h. Reducing oxygen from 20% to just 15 or 10% induces an increase in VEGF message. Maximal induction is seen at 5, 2 or 1% O{dollar}\sb2{dollar}.;Apart from the female reproductive system, neovascularization in adults is essentially limited to disease states. Tumor growth, characterized by the development of hypoxic regions, is angiogenesis-dependent. VEGF is secreted at high levels by tumors of various origins. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric protein known to be a transcriptional activator of the human erythropoietin gene in hypoxic cells. We show the involvement of HIF-1 in activating VEGF transcription in Hep3B cells. VEGF sequences mediated transcriptional activation of luciferase (luc) reporter gene expression in response to hypoxia in Hep3B cells. A 47-base pair (bp) sequence, located 985 to 939 bp upstream of the VEGF transcription initiation site, mediated hypoxia-inducible gene expression. Reporters containing VEGF sequences, in the context of either the native VEGF or the heterologous SV40 promoter, were co-transfected with expression vectors encoding HIF-1{dollar}\alpha{dollar} and HIF-1{dollar}\beta{dollar}. Luc expression was significantly increased in both hypoxic and non-hypoxic cells relative to cells transfected with reporters alone. A HIF-1 binding site was identified in the 47-bp hypoxia response element. A 3-bp substitution eliminated HIF-1 mediated transcriptional activation in response to hypoxia and/or recombinant HIF-1. Co-transfection of a luc reporter containing the 47-bp element with a vector expressing a dominant negative form of HIF-1{dollar}\alpha{dollar} inhibited transcriptional activation in hypoxic cells in a dose-dependent manner. These data indicate that VEGF expression responds rapidly to a reduction in oxygen and that this response is partly due to an increase in transcription mediated by HIF-1.
    • Steroid control of growth factor gene expression in the rat uterus: The role of vascular endothelial growth/permeability factor in the regulation of estrogen-induced increases in uterine vascular permeability and growth

      Cullinan-Bove, Kathleen; Koos, Robert D. (1995)
      In the uterus, estrogen from the ovary stimulates microvascular permeability and edema, followed by proliferation of both epithelial cells and cells of the richly vascular stroma. These responses can be mimicked in an ovariectomized (ovx) animal by administering 17{dollar}\beta{dollar}-estradiol (E{dollar}\sb2{dollar}). Uterine hypertrophy and hyperplasia may be the result of an E{dollar}\sb2{dollar}-induced increase in locally produced growth factors. This dissertation investigates the expression of several growth factors in the uterus by E{dollar}\sb2{dollar}, and correlates expression patterns of these growth factors with the E{dollar}\sb2{dollar}-induced uterotropic responses. Using the specific and sensitive method of reverse transcription-polymerase chain reaction (RT-PCR), we have shown that E{dollar}\sb2{dollar} administered to immature, ovx animals regulates the expression of several growth factors in a time dependent manner. mRNA for acidic fibroblast growth factor (aFGF), basic FGF (bFGF), keratinocyte GF (KGF) and transforming GF-beta{dollar}\sb2{dollar} (TGF-{dollar}\beta\sb2{dollar}) are all expressed in the rat uterus and their expression is enhanced by E{dollar}\sb2{dollar} treatment (6 h). Also, E{dollar}\sb2{dollar} regulation of mRNA levels for vascular endothelial growth/permeability factor (VEG/PF) was investigated. As early as 30 minutes after E{dollar}\sb2{dollar} treatment, mRNA levels for VEG/PF begin to increase and reach maximal levels at 2 h (8-fold higher than control levels). Preliminary studies show that following E{dollar}\sb2{dollar}-treatment, VEG/PF mRNA levels are highest in uterine epithelial cells. Metabolic labelling and immunoprecipitation have shown that E{dollar}\sb2{dollar} stimulates a corresponding increase in uterine VEG/PF protein production. The increase in VEG/PF levels immediately precedes the known increase in E{dollar}\sb2{dollar}-induced uterine water imbibition, and suggests that VEG/PF, a potent stimulator of vessel permeability, might play a role in this uterotropic response. The interaction of E{dollar}\sb2{dollar} and progesterone (P{dollar}\sb4{dollar}) on the regulation of VEG/PF and bFGF expression in the immature, ovx animal was also investigated. Initial studies showed that treatment of immature, ovx rats with P{dollar}\sb4{dollar} alone stimulated the expression of VEG/PF similar to the effect of E{dollar}\sb2{dollar}. However, when P{dollar}\sb4{dollar} and E{dollar}\sb2{dollar} were administered together, after 48 h of E{dollar}\sb2{dollar}-priming, P{dollar}\sb4{dollar} suppressed the E{dollar}\sb2{dollar}-induced increase in VEG/PF mRNA but had no effect on the E{dollar}\sb2{dollar}-induced increase in bFGF mRNA. Finally, the ability of recombinant human VEG/PF to mimic the effect of E{dollar}\sb2{dollar} in increasing uterine vessel permeability by direct injection into the uterine lumen was investigated. Similarly we attempted to inhibit the effects of E{dollar}\sb2{dollar} by treating the animals, intraluminally, with antisense VEG/PF and anti-VEG/PF antiserum. These studies were not successful. The E{dollar}\sb2{dollar}-induced uterine proliferative response most likely involves a complex interaction of a variety of growth factors. The data have shown that E{dollar}\sb2{dollar} stimulates expression of several growth factors in the rat uterus. The rapid E{dollar}\sb2{dollar}-induced increase in VEG/PF mRNA (2 h), suggests that VEG/PF expression may be a prerequisite for the subsequent expression or action of other growth factors present in the uterus.