Angiotensin II (ANG II) is known to play an important physiological role in the blood pressure and body fluid homeostasis mainly via its subtype 1 (AT1) receptors. Chronic infusions of angiotensin II at doses that are initially subpressor can enhance the pressor response to angiotensin II and lead to a slowly developing pressor effect, elevations of intrarenal angiotensin II levels and reductions in renal renin content and renin mRNA. The elevated circulating and intrarenal angiotensin II levels exert substantial influences on renal hemodynamics and excretory function and prevent the kidney from exerting its antihypertensive action via pressure-diuresis and -natriuresis function. Thus, the present study was designed to test the hypothesis that chronic infusion of subpressor doses of angiotensin II causes changes in proximal tubular angiotensin II concentrations, renal hemodynamic and excretory function that can be reversed by blockade of AT1 receptors. The blood pressure and renal functional responses to chronic and acute blockade of AT1 receptors by losartan were evaluated in ANG II-infused hypertensive rats. The autoregulatory capability of renal blood flow (RBF) and glomerular filtration rate (GFR) and renal arterial pressure-natriuresis relationships were assessed in ANG II-infused rats with and without chronic losartan treatment. The intrarenal and proximal tubular angiotensin II concentrations were also measured in ANG II-induced hypertensive rats. ANG II-induced hypertension was associated with a reduced GFR which could not be completely restored by acute losartan administration. In contrast, chronic losartan treatment prevented the decreases in GFR along with the increases in arterial pressure. The autoregulatory capability of RBF and GFR, but not medullary blood flow (MBF), was impaired in ANG II-infused hypertensive rats. The renal arterial pressure-natriuresis and -diuresis curves were shifted to the right. Chronic losartan treatment prevented the impairment of autoregulation of RBIF and GFR, and improved the pressure-natriuresis and -diuresis relationships. In spite of the hypertension, the proximal tubular fluid angiotensin II concentrations in ANG II-infused rats were not significantly different from hose found in normotensive control rats. These data suggest that chronic angiotensin II infusions lead to alterations in renal hemodynamics and excretory function, impaired autoregulation of RBF and GFR, but not MBF, and a rightward shift of the pressure-natriuresis relationship. These renal function changes contribute to the development of hypertension of this model / acase@tulane.edu

Cloned A-channel candidates (Kv1.4, 3.4, 4.1, 4.2 and 4.3) were initially expressed in Xenopus oocytes. However, functional discrepancies exist between native A-currents and currents resulting from Kv alpha-subunit expression in oocytes. The discrepancies may stem from heteromeric subunit assembly or may simply result from the different expression environments. Therefore, we compared native A-current (IA) from adult rat sympathetic neurons with those from cloned A-channel candidates expressed in HEK293T cells. Native and cloned IA were recorded under similar experimental conditions using the patch-clamp technique. IA of SCG neurons activated at more hyperpolarized voltages than cloned IA. Inactivation of native IA occurred at more negative potentials and the recovery from inactivation was more rapid than that of cloned IA. Native current was more sensitive to 4-aminopyridine than currents from the Kv 4 family. These results demonstrate that single cloned Kv-channel alpha-subunits do not duplicate the native A-current of sympathetic neurons M-channels can be modulated by a large array of Gq/11-coupled receptors. The histamine H1 receptor (H1R) also appears to be a Gq/11-coupled receptor, however, the involvement of H 1R in M-channel modulation has not been addressed. Therefore, we investigated if histamine could inhibit recombinant M-channels produced by KCNQ2/KCNQ3 channel subunit expression via H1R receptors expressed in HEK 293T and HeLa cells. Our studies showed that activation of H1R by histamine significantly inhibited recombinant M-currents (IM). This inhibition was not a peculiarity of the HEK 293T cell since IM inhibition by histamine also occurred in HeLa cells. Activation of a pertussis toxin-insensitive G protein was involved in histamine inhibition. Moreover, Galphaq/11 is a mediator of the inhibition since expression of the Galpha q/11 buffer significantly prevented histamine inhibition. However, expression of Gbetagamma buffers also strongly attenuated IM inhibition by histamine, indicating that the Gbetagamma dimer was also involved in IM modulation by histamine. The histamine inhibition of I M was not mediated by Ca2+ release from an intracellular Ca2+ store since thapsigargin pretreatment with could not prevent histamine inhibition. Pretreatment with nonspecific protein kinases inhibitors were also unable to prevent histamine inhibition, indicating activation of protein kinases was not involved in histamine inhibition of IM / acase@tulane.edu

The blood-brain barrier describes characteristics unique to the cerebral microvasculature. Together, these components regulate the passage of materials between the central nervous system and the periphery. Some material are able to penetrate the vascular barrier by nonsaturable processes such as passive diffusion and leakage. Saturable transport also serves as a mechanism for trafficking compounds, including certain peptides and proteins, between the blood and brain In this work, the fates of peptides and proteins relative to the blood-brain barrier were investigated after intravenous (IV) and intracerebroventicular (ICV) injection. A detailed autoradiographic investigation of distribution within the brain of radioiodinated substances, including lyrosine-MSH release-inhibiting factor-1 (Tyr-MIF-1), after ICV injection in rats was conducted. Rates of disappearance from the central nervous system (CNS) for the injected compounds were also determined by computer-assisted image analysis of the autoradiographic images. The unique patterns of distribution within the brain and disappearance from the CNS were compared for each of the compounds The ability of blood-borne amyloid $\beta$-protein, a compound implicated in the onset of Alzheimer's disease, to cross the BBB was also assessed. Amyloid $\beta$-protein accumulated in the brains of mice by a nonsaturable mechanism after IV administration. Amyloid $\beta$-protein was shown to have a high degree of association with the brain capillaries. Furthermore, intact amyloid $\beta$-protein was identified in cerebrospinal fluid (CSF) and cortical homogenates by acid precipitation, high performance liquid chromatography (HPLC) analysis, and gel electrophoresis The consequences of the saturable transport of blood-borne neuroimmunomodulatory cytokines, including interleukin (Il)-1$\alpha,$ into the parenchymal space of the brain was studied as well. Film autoradiography was used to demonstrate cytokine transport and sites of accumulation in the brain after IV administration in mice. Specific localization of Il-1$\alpha$ was detected within the caudal region of the septal nuclei Together, these observations describe processes involved in the transport of peptides and proteins across the blood-brain barrier and their distribution within the CNS, as well as assisting in identifying the ability of toxic compounds to penetrate the barrier. Such information may be useful in the prevention of certain neuropathologies. A fuller understanding of the routes of passage for proteins and peptides across the barrier elucidated with these methodologies may lead to their use as delivery systems for therapeutic agents / acase@tulane.edu

The Anterior Commissural Nucleus (ACN) is an hypothalamic accessory nucleus that has been implicated in the expression of maternal behavior. Estrogen increases oxytocin (OT) mRNA levels in the ACN. However, these ACN OT neurons have not been shown to possess estrogen receptors (ER), nor to concentrate $\sp3$H-estradiol. Since these OT neurons are in close proximity to a group of ER-containing cells has led to speculation that an indirect pathway exists for estrogen activation of the ACN OT neurons. In the present study, several of the effects of estrogen on the OT neurons of the ACN were examined, and potential neuronal links between ER-containing cells and OT neurons were explored Using double-labeling immunocytochemistry (ICC), we confirmed that ER was not present in the OT neurons of the ACN. Four daily injections of EB, in comparison to a similar injection schedule with oil, increased by 26% the number of OT-ir neurons (p $<$ 0.001) and by 40% their staining intensity (p $<$ 0.03). A comparison of the distribution of ACN OT-ir neurons in EB- and oil-injected animals suggested a greater increase in cell number in the more rostral areas of the ACN. Eleven neuroactive substances were examined using ICC and in situ hybridization methods to identify the type of neuron in the ACN that might co-localize ER. Dopamine-beta-hydroxylase, neuropeptide Y, tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) were found in the ACN. Only TH and GAD were found in cell bodies. In situ hybridization for GAD mRNA suggested that GAD is present in many of the ER-containing neurons in the ACN. ICC studies confirm that the OT neurons of the ACN co-localize GABA receptor on their membranes. Pseudo-confocal microscopy showed that estrogen treatment resulted in a decrease of glial coverage from 80% of OT neurons examined to 20% (p $<$ 0.05). It was concluded that the glia and OT neurons of the ACN are responsive to estrogen, and that the response of the OT neurons is mediated at least in part by the ER-containing, GABAergic neurons of the ACN / acase@tulane.edu

This study was designed to determine the structural requirements for activation of angiotensin receptors and to investigate regional differences in angiotensin responses in the systemic circulation. The effect of local modulators such as cyclooxygenase products and nitric oxide release on responses to the angiotensin peptide were investigated. The pharmacologic properties of the nonpeptide angiotensin II type 1 receptor antagonists were determined in the feline systemic vascular bed In order to control for differences due to flow changes and to determine the direct effect of the angiotensin peptides in the systemic vascular bed, the peptides were directly injected into the regional vascular bed in which blood flow was maintained constant with a pump The data presented in this dissertation show that angiotensin II is the most potent of the endogenous vasoconstrictors studied in the feline hindquarters vascular bed. Ang III, the C-terminal heptapeptide metabolite of angiotensin II was also very potent, having approximately 75-95% of the activity of angiotensin II. Angiotensin 1-7, an important brain peptide and angiotensin 3-8 had very weak vasoconstrictor activity however, Ang 1-7 was shown to exhibit vasodilator activity which was dependent on dose and regional vascular bed studied. The dilator responses to angiotensin 1-7 were mediated in part by nitric oxide release in the mesentery and hindquarters vascular beds because they were partially inhibited by the nitric oxide synthase inhibitor, L-NAME. The vasoconstrictor effects of all the angiotensin peptides studied were not modulated by the release of cyclooxygenase products. This study shows 50% upstream conversion of angiotensin I to angiotensin II in the hindquarters and mesenteric vascular beds by the use of the angiotensin-converting enzyme inhibitors captopril, enalaprilat and ramiprilat. The present data also demonstrates that the nonpeptide antagonists, DuP 753 and EXP 3174 are highly selective and potent antagonists of the pressor responses of all the angiotensin peptides studied. There is neither a biphasic pattern of inhibition of Ang II responses by DuP 753 nor an intensification of blockade in the cat. These data also provide support for the hypothesis that spare angiotensin II receptors are present in resistance vessel elements in the hindquarters vascular bed of the cat In summary, the contributions of the present studies to the current literature on the pharmacology of the angiotensin peptides are firstly, Ang II is a very potent vasoconstrictor in the skeletal vascular bed of the cat; secondly, this is the first documentation of a vasodilator response to angiotensin 1-7 mediated by nitric oxide release, and finally with the use of the highly selective nonpeptide angiotensin receptor antagonists we have demonstrated the existence of spare angiotensin receptors in the hindquarters vascular bed of the cat. (Abstract shortened by UMI.) / acase@tulane.edu

This study was designed to investigate the responses of bradykinin and related peptides in the pulmonary circulation. The pulmonary vascular bed exhibits a great ability to adapt to general and local changes in blood flow, and regulation of the pulmonary circulation is probably localized. The effect of local modulators, such as cyclooxygenase products, nitric oxide and the putative endothelium-dependent hyperolarizing factor on responses to bradykinin and related peptides were investigated. The pharmacologic properties of the new kinin receptor antagonist, HOE-140, were also investigated under conditions of controlled blood flow and constant left atrial pressure in the intact-chest cat These studies demonstrate that bradykinin is a potent vasodilator when lobar arterial pressure is raised to a high steady level with an infusion of U-46619, a thromboxane A$\sp2$ mimetic. Following administration of HOE-140, a kinin B$\sp2$ receptor antagonist, vasodilator responses to bradykinin were reduced in a selective manner. Vasodilator responses to bradykinin were not dependent on activation of muscarinic receptors, K$\sp{+}\sb{\rm ATP}$ channels, the release of vasodilator prostaglandins, or changes in bronchomotor tone. The results suggest that vasodilator responses to bradykinin occur through activation of kinin B$\sp2$ receptors, which mediate nitric oxide release. Nitric oxide subsequently activates soluble guanylate cyclase and increases guanosine 3$\sp\prime, 5\sp\prime$-cyclic monphosphate levels in the pulmonary vascular bed The data investigated the role of K$\sp{+}\sb{\rm ATP}$ channel activation and nitric oxide synthase inhibitors in the pulmonary vascular bed. The results involving inhibition of K$\sp{+}\sb{\rm ATP}$ channels show that U-3788A (nonsulfonylura) and glybenclamide (sulfonylurea), agents which are extensively used in the study of vascular smooth muscle K${+}\sb{\rm APT}$ channnel mechanisms and attenuate vasodilator responses to K$\sp{+}\sb{\rm ATP}$ channel openers, have pronounced effects on thromboxane/prostaglandin receptor-mediated vasoconstrictor responses in the pulmonary vascular bed of the cat. N$\sp\omega$-nitro-L-arginine, N$\sp\omega$-nitro-L-arginine methyl ester, N$\sp\omega$-nitro-L-arginine benzyl ester, and L-N$\sp5$-(1-iminoethyl) ornithine had similar inhibitory effects on bradykinin and substance P. The data suggest that these L-arginine analogs are useful probes for studying nitric oxide mediated responses in the pulmonary vascular bed and the alkyl esters of N$\sp\omega$-nitro-L-arginine do not block muscarinic receptors in the cat. Furthermore, L-N$\sp5$-(1-iminoethyl) ornithine did not increase lobar and systemic arterial pressure in comparison to the other nitric oxide synthase inhibitors, suggesting that basal release and endothelium-dependent agonist-stimulated release of nitric oxide can be differentiated by the adminstration of L-N$\sp5$-(1-iminoethyl) ornithine in the pulmonary vascular bed of the cat The data also demonstrate that des-Arg$\sp9$-bradykinin produces a vasoconstrictor response under low tone conditions and that these responses are mediated by kinin B$\sb1$ receptors, the release of catecholamines within the lung and the activation of alpha adrenergic receptors. While in the same experiments when tone is raised with an infusion of U-46619, des-Arg$\sp9$-bradykinin produces a vasodilator response that is mediated by the activation of kinin B$\sb1$ receptors and the release of nitric oxide from the endothelium. These data provide pharmaclogic evidence for the existence of functionally active kinin B$\sb1$ receptors that mediate tone-dependent vasoconsrictor and vasodilator responses in the pulmonary vascular bed of the cat. (Abstract shortened by UMI.) / acase@tulane.edu

Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is a naturally occurring nonapeptide released from substrate kininogen, by the serine protease kallikrein in both tissue and plasma during tissue injury and inflammation. The peptide is rapidly inactivated in the lung by kininase II, a dipeptidyl carboxypeptidase and other peptidases. Kininase II has been shown to be identical to angiotensin I-converting enzyme which produces the potent vasoconstrictor angiotensin II from angiotensin I Kinin receptors are classically divided into B$\sb1$ and B$\sb2$ receptor subtypes based on the activities of selective agonists. Kinin B$\sb1$ receptors are activated by des-Arg$\sp9$-bradykinin, located on vascular smooth muscle cells and are reported to mediate relaxation and contraction in the dog and rabbit via the release of prostaglandins. The role of the B$\sb1$ receptor is not well understood, and this receptor subtype may not be present under normal physiologic conditions. Although B$\sb1$ receptors have been shown to be induced following noxious stimuli in the rabbit carotid artery, recent studies in the dog systemic vascular bed and isolated renal artery have demonstrated B$\sb1$ receptor mediated vasodilation under physiologic conditions. The present study will attempt to characterize the kinin peptides in a single species within two vascular beds. Most of the biological activity of bradykinin is thought to be mediated by the B$\sb2$ receptor, including vasodilation, pain, inflammation, bronchoconstriction and increased vascular permeability These studies of the kinin peptides, bradykinin, kallidin (Lys-BK), des-Arg$\sp9$-bradykinin, des-Arg$\sp{10}$-kallidin, and T-kinin (Ile-Ser-BK) were designed to address the following specific aims: (1) to study the receptor subtype mediating responses to the kinin peptides in the hindquarters and mesenteric vascular beds of the cat. (2) to study the effect of locally derived factors such as nitric oxide and prostaglandins on responses to the kinin peptides in the regional vascular beds. (3) to study the effect of angiotensin-converting enzyme inhibitors on responses to bradykinin in the regional vascular beds. (4) to characterize reactive vasodilator responses to brief periods of occlusion under natural and controlled flow conditions and the mechanism of action of reactive vasodilator responses The results of these studies demonstrate that responses to bradykinin, kallidin and T-kinin are mediated by the activation of kinin B$\sb2$ receptors coupled to the release of nitric oxide. The results of these studies also demonstrate that angiotensin-converting enzyme activity in the hindlimb vascular bed occurs upstream at or near the site of action of bradykinin. The results also demonstrate that des-Arg$\sp9$-bradykinin and des-Arg$\sp{10}$kallidin produce vasodilator responses mediated by kinin B$\sb1$ receptors under normal physiologic conditions and that these B$\sb1$ receptors are coupled to the release of nitric oxide. This study demonstrates that reactive hyperemic responses to brief periods of occlusion are significantly reduced following K$\sp{+}$ATP channel blockade and suggest that the reactive vasodilator response in the hindquarters vascular bed of the cat is dependent in part on the opening of K$\sp{+}$ATP channels / acase@tulane.edu

At least four potassium currents are believed to contribute to the repolarization phase of the action potential in human heart, including the transient outward current (Ito), the delayed rectifier (IK), ultrarapid (IKur) and inward rectifier (IK1) currents (Firek and Giles, 1995; Ravens et al., 1996). Ito recently emerged as an important current of the human heart, contributing to phase 1 repolarization of the action potential (Escande et al., 1985; Shibata et al., 1989). Studies on cloned 'Ito-like' channels have suggested that the changes in external potassium concentration may affect the properties of the channels, such as current density, channel conductance and voltage dependence and kinetics of channel gating (Demo and Yellen, 1991; Pardo et al., 1992; Tseng and Tseng-Crank, 1992). The effects of changes in external potassium concentrations on the properties of the human atrial transient outward potassium current were therefore examined in single human cardiac myocytes isolated from right atrial appendages using the whole cell variant of the patch clamp technique. We found that the modulation of human atrial Ito by extracellular potassium is different from that observed for published findings on cloned Ito-like channels. Recent studies documented the existence of significant developmental changes in the characteristics of human atrial Ito under physiologic conditions, (Crumb et 1995a). This study reports that the potassium selectivity of the transient outward channel undergoes a significant age-related change, which is reflected in a more hyperpolarized reversal potential for Ito in adult compared to pediatric cells. This developmental difference in the selectivity of Ito for K is likely to contribute to the reported developmental difference in the amplitude of this current in human atrium (Crumb et al., 1995a) We also investigated the effects of age on quinidine's interactions with human atrial potassium channels. Quinidine was selected because it is still one of the most commonly used drugs in the United States for the treatment of atrial rhythm disturbances (Grace and Camm, 1998). Furthermore, age-related changes in the cardiac actions of quinidine on the action potential duration and affinity for potassium channels have been reported in several mammalian species (Morikawa and Rosen, 1986; Wu et al., 1994). Our results indicate that there are significant developmental changes in the effects of quinidine on human atrial Ito. The major differences in drug effects include age-related changes in both the IC50 for quinidine blockade of Ito, as well as an age-related change in the mechanism of channel block and unblocking. Another major conclusion of this work is that quinidine inhibits both adult and pediatric IK1 and I Kur in an age-independent manner, while the non-selective current (I ns), which contributes to the sustained current, is insensitive to quinidine / acase@tulane.edu

Purkinje-ventricular interactions are important determinants of both normal and abnormal cardiac rhythms. These interactions are defined by two factors: (i) the unique structure of the Purkinje-ventricular junction (PVJ), and (ii) differences in the membrane ionic currents governing Purkinje (P) and ventricular (V) action potential configuration (membrane-level differences). I developed a new approach to investigate the contributions of membrane-level differences to conduction and modulation of repolarization at the PV interface. I isolated P and V myocytes from rabbit hearts and used an electronic circuit to supply the coupling current that would normally flow between the cells if they were physically coupled. This approach allowed me to record action potentials before and after coupling and to vary the junctional resistance $(R\sb{j})$ imposed between the cells. Studies completed with the PV cell pairs showed that intrinsic phase 1 repolarization largely determined directional differences in conduction. This was significant because unidirectional (P to V) block is typically attributed to the structure of the PVJ. Further, the large phase 1 repolarization intrinsic to rabbit P cells caused shortening of the V action potential (AP) upon coupling. This was significant because coupling regions with differences in intrinsic AP duration typically results in shortening of the intrinsically longer AP and prolongation of the intrinsically shorter AP. I additionally used a computational model to describe structural aspects governing source-sink relations at the PVJ. The model results verified that membrane-level differences importantly affected conduction and modulation of repolarization in a more macroscopic representation of PV interactions / acase@tulane.edu

Crustaceans, like many other invertebrates and some vertebrates, are capable of altering their color by altering the degree of pigment concentration or dispersion in specialized integumental cells known as chromatophores. Pigment translocation in the fiddler crab, Uca pugilator, is regulated by hormones released from the animal's eyestalk neuroendocrine gland, the X-organ-sinus gland complex. This complex contains no fewer than four pigment effector hormones. The release of these hormones is thought to be regulated by neurons impinging directly on the secretory cells, or on cells somewhere along the chain of neurons leading to the X-organ-sinus gland (Fingerman, 1985) Biogenic amines, including dopamine, octopamine, 5-hydroxytryptamine, and norepinephrine, have been shown to have hormone-releasing effects when injected into U. pugilator. Whether or not endogenous stores of these compounds act as neurotransmitters, however, has not been fully illustrated In the following work, I have documented the presence of dopamine, octopamine, norepinephrine and 5-hydroxytryptamine in various tissues of U. pugilator. In addition, I have provided evidence for a mechanism of synthesis of one (5-hydroxytryptamine) using pharmacological blockers against enzymes involved in its synthesis. I have also demonstrated a mechanism whereby the action of one (norepinephrine) is terminated by re-uptake and metabolism of this putitive neurotransmitter. In the final chapter, the relationship of biogenic amines and eyestalk neuropeptides, was examined / acase@tulane.edu