The effects of xylazine and alpha-adrenoreceptor antagonists on bovine uterine contractility in vitro

The effects of alpha-adrenoreceptor antagonists prazosin (alpha-1), yohimbine (alpha-2), and idazoxan (alpha-2) on xylazine-induced bovine uterine contractility were tested in vitro. Uterine strips from proestrous/estrous and diestrous cows were mounted in tissue baths containing Tyrode's solution. Changes in uterine contractility were measured by strain gauge. The following results were observed: 1) Xylazine increased uterine contractility in a dose dependent manner (cumulative concentrations: 10(-8), 3x10(-8), 10(-7), 3x10(-7) and 10(-6)M). 2) Idazoxan (10(-8), 10(-7) and 10(-6)M) and yohimbine (10(-6), 10(-5) and 10(-4)M) antagonized uterine contractility induced by xylazine in a dose-dependent manner. Idazoxan was approximately 50 to 100 times more potent than yohimbine. 3) Prazosin (10(-5)M) did not alter the effect of xylazine on uterine contractility. These results suggested that xylazine-induced uterine contractility in the cyclic cow is directly mediated by myometrial alpha-2 adrenoreceptors.     

Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1-tilting adjuvant for recombinant gD2 protein vaccination against genital herpes

Although sexually transmitted pathogens are capable of inducing pathogen-specific immune responses, vaginal administration of nonreplicating antigens elicits only weak, nondisseminating immune responses. The present study was undertaken to examine the potential of CpG-containing oligodeoxynucleotide (CpG ODN) for induction of chemokine responses in the genital tract mucosa and also as a vaginal adjuvant in combination with glycoprotein D of herpes simplex virus type 2 (HSV-2) for induction of antigen-specific immune responses. We found that a single intravaginal administration of CpG ODN in mice stimulates a rapid and potent response of CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES as well as of CXC chemokines MIP-2 and IP-10 in the vagina and/or the genital lymph nodes. Importantly, intravaginal vaccination with recombinant gD2 in combination with CpG ODN gave rise to a strong antigen-specific Th1-like immune response in the genital lymph nodes as well as the spleens of the vaccinated mice. Further, such an immunization scheme conferred both systemic and mucosal immunoglobulin G antibody responses as well as protection against an otherwise lethal vaginal challenge with HSV-2. These results illustrate the potential of CpG ODN for induction of potent chemokine responses in the genital tract and also as a vaginal adjuvant for generation of Th1-type mucosal and systemic immune responses towards a nonreplicating antigen derived from a sexually transmitted pathogen. These data have implications for the development of a mucosal vaccine against genital herpes and possibly other sexually transmitted diseases.     

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

The characteristics of adrenoceptors in homogenates of human cerebral cortex labelled by (3H)-rauwolscine

The alpha-2-adrenoceptor antagonist (³H)-rauwolscine has been used to label adrenoreptors in membranes from human cerebral cortex. The radioligand binds with high affinity (K_D 2.08 nM) to a single population of sites with a density of 135 fmoles/mg protein. Adrenoceptor antagonists displaced binding in a simple monomolecular fashion with an order of affinity rauwolscine > yohimbine > phentolamine > corynanthine > prazosin indicating binding to alpha-2-adrenoceptors. Agonists displaced with an order of affinity clonidine > (-) adrenaline > (-) noradrenaline > dopamine > (-) isoprenaline but all displayed apparent Hill coefficients less than unity indicating heterogeneity of binding. The relatively high affinity of the alpha-1 antagonist prazosin for (³H)-rauwolscine binding sites in rat cerebral cortex was not observed in the human tissue which had pharmacological properties similar to those described previously in human platelet.     

Adrenoceptor mechanisms in the cardiovascular effects of cocaine in conscious squirrel monkeys.

The purpose of the current experiment was to study the role of various adrenoceptor subtypes in the cardiovascular response to cocaine in conscious squirrel monkeys. A variety of adrenoceptor antagonists were administered i.v. prior to the administration of 0.3 mg/kg cocaine (i.v.). Cocaine alone produced an increase in both blood pressure and heart rate. The non-selective alpha adrenoceptor antagonist phentolamine produced a dose-dependent antagonism of the pressor effect of cocaine, as did the alpha-1 selective antagonist prazosin. The alpha-2 selective antagonist yohimbine had no effect on the pressor effect of cocaine. The non-selective beta antagonist propranolol enhanced the pressor effect of cocaine as did the beta-1 selective antagonist atenolol. However, the effect of atenolol was not dose-dependent. The beta-2 selective antagonist ICI 118,551 and labetalol, which blocks both alpha and beta adrenoceptors, did not alter the pressor effect of cocaine. Propranolol, atenolol, and labetalol all antagonized the tachycardiac effect of cocaine in a dose-dependent manner, while the beta-2 antagonist ICI 118,551 did not. Phentolamine, prazosin and yohimbine also reduced the tachycardiac effect of cocaine, although these effects were dose-dependent only for yohimbine, which also significantly elevated baseline heart rate. These results indicate that alpha-1 adrenoceptor mechanisms mediate the pressor effect of cocaine, while beta-1 adrenoceptor mechanisms are involved in the tachycardiac effect of cocaine in squirrel monkeys. Propranolol potentiated cocaine's pressor effect through beta-2 independent mechanisms. Thus, neither alpha-2 nor beta-2 adrenoceptor mechanisms appear to be involved in cocaine's cardiovascular effects.     

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.     

Nanoparticle Transport from Mouse Vagina to Adjacent Lymph Nodes

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.     

Comparative effects of rauwolscine, prazosin, and phentolamine on blood pressure and cardiac output in anesthetized rats

The endogenous role of the alpha-adrenergic system in the maintenance of mean arterial pressure (MAP), total peripheral resistance (TPR), cardiac output (CO) and its distribution, and plasma norepinephrine and epinephrine release was investigated by the administration of selective alpha-adrenoceptor antagonists to halothane-anesthetized rats. The blockade of alpha 1-, alpha 2-, and both alpha 1- and alpha 2-receptors was accomplished by i.v. infusions of prazosin, rauwolscine, and phentolamine, respectively. The microsphere technique was used for the determination of CO and its distribution. Since the infusions of the three antagonists caused similar decreases of MAP and heart rate, the results suggest that postjunctional alpha 1- and alpha 2-receptors are both important in the control of MAP. During the infusion of prazosin, TPR was decreased but CO was not changed. In contrast, CO was decreased but TPR was not changed during the infusions of rauwolscine and phentolamine. Thus, CO was reduced after the blockade of alpha 2- but not alpha 1-receptors. All three antagonists caused an increase in percent distribution of CO to the lungs and muscle, suggesting that the sympathetic nervous system plays the greatest vasoconstrictor influence in the lungs and muscle via stimulations of both subtypes of alpha-adrenoceptors. The administration of either prazosin or rauwolscine caused little change in plasma catecholamine levels. In contrast, phentolamine caused large increases in both epinephrine and norepinephrine levels. Therefore catecholamine release was only increased after concurrent blockade of both alpha 1- and alpha 2-adrenoceptors.     

Adrenergic stimulation of inositol-phosphate production in a genital tract smooth muscle cell line

Catecholamines are important in the modulation of smooth muscle contractile activity; this study was undertaken to evaluate adrenoceptor stimulation of intracellular inositol-phosphate production in a genital tract smooth muscle myocyte. DDT1 MF-2 smooth muscle myocytes, derived from a hamster ductus deferens leiomyosarcoma, were loaded with 3H-inositol, incubated in 10 mM LiCl, then stimulated with adrenergic agonists with and without antagonists. Subsequently, the inositol phosphates were isolated by anion-exchange chromatography. In the presence of norepinephrine (NE), inositol trisphosphate (IP3) was produced by 30 s and peaked at 2 min; inositol 1-phosphate was also apparent by 30 s, and continued to increase over 15 min. Clonidine (an alpha-2 agonist), isoproterenol, and NE in the presence of phentolamine or prazosin (an alpha-1 antagonist) failed to increase IP3. In contrast, NE in the presence of yohimbine (an alpha-2 antagonist) or propranolol stimulated IP3 production to levels comparable to that stimulated by NE alone. These studies provide evidence that inositol phosphate production is involved in alpha-1 adrenergic signal transduction in DDT1 MF-2 myocyte.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

The cardiac effects of arginine vasotocin in amphibians

1. The cardiac effects of arginine vasotocin (AVT) on isolated atria were examined in three anuran and one urodele species. 2. AVT produced dose-related positive chronotropic and inotropic responses. 3. The responsiveness of the atrial tissue varied among species. 4. Both the basal atrial rate (AR) and tension (T) were attenuated in the presence of phentolamine and propranolol, alpha- and beta-adrenergic antagonists. Isoproterenol, a beta-adrenergic agonist, increased both AR and T, an effect which would be inhibited by propranolol. 5. The effects of AVT on both AR and T were not inhibited by alpha- and beta-adrenergic blockers, nor by verapamil and imidazole with the dosages used in the present study. 6. On the contrary, the effects of AVT on AR, but not T, was enhanced in the presence of both alpha- and beta-adrenergic blockers. 7. The mechanism of action of AVT on the amphibian atrium remains unknown.     

Effect of moxisylyte hydrochloride on isolated human penile corpus cavernosum tissue

The effect of moxisylyte hydrochloride on isolated human penile corpus cavernosum tissue was investigated and compared with other a-adrenergic antagonists. Moxisylyte produced a concentration-dependent relaxation of a norepinephrine-induced (1 x 10(-5) M) contraction of the corpus cavernosum tissue. Pretreatment with 1 x 10(-6) M doses of moxisylyte reduced competitively the norepinephrine-induced contraction. The competitive effect of prazosin was strongest among four tested agents, followed by phentolamine, moxisylyte, and then yohimbine. The activity ratio for each antagonist is 2.4 for moxisylyte, as compared with 28.2 for prazosin, 6.7 for phentolamine, and 1.6 for yohimbine respectively. Moxisylyte hydrochloride is an agent with potential clinical and research uses capable of producing erection when injected intra-cavernously.     

Propranolol antagonizes hypotension induced by alpha-blockers but not by sodium nitroprusside or methacholine

A pressor response has been observed with propranolol, a nonselective beta-adrenoceptor antagonist, in animals given a nonselective alpha-adrenoceptor antagonist. This study investigates whether a pressor response to propranolol occurs in conscious unrestrained rats following a hypotensive response induced by phentolamine (nonselective alpha-antagonist), prazosin (selective alpha 1-antagonist) and (or) rauwolscine (selective alpha 2-antagonist), sodium nitroprusside (smooth muscle relaxant), or methacholine (muscarinic agonist). The rats were subjected to a continuous infusion of a hypotensive agent or normal saline followed by i.v. injection of propranolol. The infusion of phentolamine significantly decreased mean arterial pressure (MAP). Subsequent injection of propranolol restored MAP to the control level. Prazosin and rauwolscine each caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a significant decrease in MAP. Subsequent injection of propranolol caused a large pressor response which increased MAP to 20% above control MAP prior to the administration of drugs. Nitroprusside or methacholine each caused a significant decrease in MAP, but the hypotension was not antagonized by propranolol. The concurrent infusions of a low dose of nitroprusside and prazosin caused a significant decrease in MAP which was reversed by propranolol. The infusion of saline did not alter MAP, and propranolol did not cause a pressor response. It is concluded that propranolol antagonizes the hypotensive effect of an alpha-blocker but not that of sodium nitroprusside or methacholine. Our results suggest the presence of a specific interaction between alpha- and beta-antagonists.     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

Xylazine enhances porcine myometrial contractility in vitro: possible involvement of alpha 2-adrenoceptors and Ca2+ channels

The effects of xylazine on porcine myometrial contractility were studied in vitro using uterine strips to determine the alpha 2-adrenergic influences during the diestrous stage of the estrous cycle. Xylazine (10(-8)-10(-5) M) caused a dose-dependent increase in the amplitude of myometrial contractility. The alpha 2-adrenoceptor antagonists idazoxan and yohimbine (10(-8)-10(-6) M) blocked the effects of xylazine in a dose-dependent manner. Yohimbine was approximately 10 times more potent than idazoxan in this regard. In contrast, an alpha 1-adrenoceptor antagonist prazosin (10(-7) and 10(-6) M) did not block the xylazine-induced increase in myometrial contractility, but a higher dose of prazosin (10(-5) M) did reduce the effects of xylazine. When the porcine uterine strips were pretreated with Ca2(+)-free Tyrod's solution or verapamil, a Ca2+ channel blocker, the effects of xylazine on myometrial contractility were completely abolished, whereas those of carbachol were only moderately reduced. The results suggest that the xylazine-induced myometrial contractility is mediated by alpha 2-adrenoceptors and that this effect is mediated, at least in part, by Ca2+ channels, whereas the effect of carbachol is attributed to an increase in both Ca2+ entry and release of Ca2+ from intracellular pools.     

Immunohistochemical localisation of androgen receptor during sex-specific morphogenesis in the fetal mouse

The distribution of androgen receptors (ARs) in paraffin serial sections of day 17 and day 18 male and female mouse embryos was investigated. In the cranial section of the genital tract AR expression was restricted to Wolffian structures while Müllerian ducts and surrounding mesenchyme were AR negative. In the fusion zone with the urogenital sinus the epithelial components of the vaginal bud were clearly distinguished by differential AR expression, which was faint in the Wolffian ducts, totally missing in the Müllerian ducts, and intense in the sinus ridges with the most intense expression in the morphogenetically active mesenchyme, indicating a new mechanism of negative control of vagina formation via androgens. Expression of ARs outside the genital tract was observed: (1) in loose interstitial mesenchyme extending into the retroperitoneal space up to the coeliac artery, indicating androgen effects during ascent of the kidneys and descent of intraperitoneal organs, (2) in the trigone of the bladder indicating androgen involvement in the development of the vesico-ureteral junction, and (3) in loose mesenchyme between striated muscle fibres and around pelvic skeletal elements, indicating mediation of androgen effects on the musculoskeletal system via loose mesenchyme.     

Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture.     

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of T_h1 and T_h17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher T_h17 and T_h1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c⁺ DCs in the vagina were the predominant APC population responsible for priming these T_h17 responses, and a potent source of IL-6 and IL-1β, important factors for T_h17 differentiation. T_h17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for T_h17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored T_h17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient T_h1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4⁺ T cell anti-viral immunity by priming vaginal DCs to induce T_h17 responses through an IL-1-dependent pathway.     

RNA-dependent protein kinase is required for alpha-1 interferon transgene-induced resistance to genital herpes simplex virus type 2

We investigated the mechanism of resistance to genital herpes simplex virus type 2 (HSV-2) infection in mice transfected with the murine alpha-1 interferon (IFN-alpha1) transgene. In situ transfection of mice with the IFN-alpha1 transgene resulted in an elevation in an IFN-responsive gene, RNA-dependent protein kinase (PKR), but not 2',5'-oligoadenylate synthetases (OAS), in vaginal tissue. Coupled with the finding that mice lacking a functional PKR pathway were no longer resistant to genital HSV-2 infection following transfection with the IFN-alpha1 transgene in comparison to wild-type mice or mice lacking a functional OAS pathway, these results suggest that PKR is the dominant antiviral pathway activated by the IFN-alpha1 transgene.