Chronic treatment with red wine modulates the purinergic neurotransmission and decreases blood pressure in hypertensive SHR and diabetic-STZ rats.

It is known that red wine has cardioprotective properties. However, its influence is unknown about purinergic system. Therefore, we study the influence of the treatment with red wine or ethanol in purinergic neurotransmission. We used Wistar Kyoto rats (WKY), diabetic streptozotocin-induced WKY and spontaneously hypertensive rats (SHR), treated with red wine (12.5%) or ethanol (12.5%). The cardiovascular function stimulated with purinergic agonists and systolic blood pressure (SBP) was assessed. In atria of diabetics and SHRs, the P1 receptor response was decreased, unlike the P2 receptor response was increased. Likewise, in aorta the affinity to adenosine (ADO) was decreased from SHRs and diabetics. Furthermore, the P2X function was increased just SHRs. All these alterations were improved after treatment with red wine, resulting in reduction of SBP from diabetics and SHRs, but not when treated with ethanol. This study has important implications, because it is shown that consumption of red wine can improve cardiovascular system by purinergic neurotransmission.

Physiopharmacological properties of the testicular capsule: A concise review.

The testicular capsules of different mammalian species exhibit spontaneous motor activity. In addition, contractions can be mediated by neuronal stimulation or exogenous drug administration. However, the physiological role of testicular capsule motor activity is still not well understood. Nevertheless, there is evidence for putative roles in spermatozoa transport from the testis to the caput epididymis, control of interstitial/intratesticular pressure and testicular blood flow. In this review, we have collated information about the agents that regulate testicular capsule motor activity, their receptors and second messengers as well as the impact of altered testicular capsule function on the male reproductive system. Furthermore, we highlight the knowledge gaps in the physiology and pharmacology of the testicular capsule as indicators of future research directions that may lead to a better understanding of the physiological role of testicular capsule motor activity and its importance in male fertility.

Functionomics: the analysis of a postgenomic concept on the basis of pregenomic pharmacological studies in smooth muscle.

The term functionomics (Amin 2003, Neumann et al. 2004) refers to a postgenomic integrated Systems Biology (Attur et al. 2002) using a multidimensional approach for cells, tissues and organs. It considers current or future involvement among genomics, proteomics or metabolomics, including the main factors that cause biological responses and modulation under different conditions. Our objective in the present review is to summarize the contemporary understanding of functionomics of smooth muscle pharmacology, based on the results obtained on the pregenomic era during several years in our laboratory. The present approach is based on the knowledge of the dynamics of the receptor system, which comprises a cascade of phenomena, leading from the drug administration to the final biological response. We will describe several conditions in which the final effect is modified, based on perturbations induced on drug absorption, distribution, metabolism, interaction with receptors and mobilization of second messengers, as well as by interactions with a second receptor system. We will also discuss the gaps that need to be fulfilled in order to obtain a clear and better understanding of the receptor system in smooth muscle, and to narrow the bridge between ourknowledge of the function of biological systems, genomics, and other recently introduced areas.

Use of transgenic (knockout) mice reveals a site distinct from the alpha2A-adrenoceptors for agmatine in the vas deferens.

The inhibitory effect of agmatine on electrically induced contractions was studied in vas deferens of Adra 2a transgenic mice lacking alpha(2A)-adrenoceptors. Agmatine and clonidine caused a concentration-dependent inhibition of twitches. However, while agmatine showed a similar pIC(50) value in control and transgenic mice, the pIC(50) value for clonidine was about 30-fold lower in knockout mice. In both strains, yohimbine shifted the curve for clonidine, but not for agmatine, even when a 100-fold higher concentration of yohimbine was employed. Our results indicate that inhibition by agmatine in mouse vas deferens is not simply due to interactions with alpha(2)-adrenoceptors in our experimental conditions.

Decreased noradrenergic and serotonergic reactivity of vas deferens of newborn rats from mothers treated with the serotonin reuptake inhibitor fluoxetine during pregnancy and breast-feeding.

Female Wistar rats were treated with the serotonin reuptake inhibitor fluoxetine (10 mg/kg/i.p/day), during pregnancy and breast-feeding, for the study of the corresponding newborn rats. At the end of the preweaning period, the 30-day old litters had their vas deferens removed for testing peripheral sympathetic reactivity, through the following experiments in vitro: (a) concentration-contraction curves for serotonin and for the adrenergic agonists noradrenaline, phenylephrine, clonidine and dopamine or for the indirect agonist tyramine (b) contractions induced by electric field stimulation, as an indicator of sympathetic neurotransmission (c) release of endogenous noradrenaline, measured by real-time determinations on HPLC (d) Ca(+2) time-contraction curves, to check for changes on Ca(+2) translocation. Our results showed that the affinity (pD(2)) for serotonin was strikingly decreased by about 1.5 log units. The pD(2) for adrenergic agonists was decreased by about 0.5 log units, except for dopamine and clonidine. The maximum effects and intrinsic activity were decreased only for dopamine. On the other hand, the response to Ca(+2) and the release of noradrenaline from nerve terminals were not modified. In additional experiments, the mother's body weights were measured, showing a decrease during gestation and a recovery during lactation while the offspring's weights were lower than controls. It is concluded that, besides the alterations on body weights, changes on noradrenergic and serotonergic mechanisms were observed and persisted in the newborn, at least one month after parturition.

Altered mitochondrial function, calcium signaling, and catecholamine release in chromaffin cells of diabetic and SHR rats.

Comorbidity of diabetes and hypertension is frequent. Here, we have performed a comparative study in three animal models namely, normotensive Wistar Kyoto (WKY) rats, streptozotocin-induced diabetic rats (STZ), and spontaneously hypertensive rats (SHR). With respect WKY rats, we have found the following alterations in adrenal chromaffin cells from STZ and SHR rats: (1) diminished Ca2+ currents; (2) augmented [Ca2+]c elevations and catecholamine release in cells stimulated with angiotensin II or high K+; (3) unchanged expression of angiotensin II receptors AT1 and AT2; (4) higher density of secretory vesicles at subplasmalemmal sites; (5) mitochondria with lower cristae density that were partially depolarized; and (6) lower whole cell ATP content. These alterations may have their origin in (i) an augmented capacity of the endoplasmic reticulum [Ca2+] store likely due to (ii) impaired mitochondrial Ca2+ uptake; (iii) augmented high-[Ca2+]c microdomains at subplasmalemmal sites secondary to augmented calcium-induce calcium release and to inositol tris-phosphate receptor mediated enhanced Ca2+ mobilization from the endoplasmic reticulum; and (iv) augmented vesicle pool. These alterations seem to be common to the two models of human hypertension here explored, STZ diabetic rats and SHR hypertensive rats.

Altered mitochondrial function, capacitative calcium entry and contractions in the aorta of hypertensive rats.

OBJECTIVE: It has been suggested that Ca entry through store-operated Ca channels (SOCs) is regulated by a dynamic interplay between the endoplasmic reticulum Ca stores and the mitochondria. These relationships drive the activation and inactivation of SOCs, yet it remains unclear whether this regulation of SOCs by mitochondria is altered in the aorta of spontaneously hypertensive rats (SHRs). METHODS: We performed a thorough study of the mitochondrial membrane potential, the ability of mitochondria to deal with cytosolic Ca, capacitative Ca entry (CCE), and stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (orai1) protein expression, as well as the contractile capacity of aortic rings, in normotensive Wistar Kyoto rats (WKYs) and SHRs. RESULTS: Changes were observed in aortic tissue and cultured vascular smooth muscle cells isolated from SHRs relative to WKYs, including more depolarized mitochondria, stronger CCE upon the addition of Ca, larger cytosolic Ca transients (cytosolic Ca concentration) or aortic ring contraction elicited by endoplasmic reticulum depletion and a significant increase in STIM1 protein expression but not of orai1. CONCLUSION: These results suggest that the impaired Ca buffering capacity of partially depolarized mitochondria dysregulates CCE, leading to overfilling of the endoplasmic reticulum Ca store through enhanced STIM1/orai1 interactions and an increase in aorta contractions in SHRs. Thus, understanding the implications of the alterations to STIM1/orai1, and their relationship to mitochondria, may aid drug development and therapeutic strategies to treat hypertension, as well as its long-term sequelae in poorly controlled patients.

Aging-related changes of intracellular Ca2+ stores and contractile response of intestinal smooth muscle.

In this study, we investigated the effect of aging on intracellular Ca2+ stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1-G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2+-activated K+ channels in coronary smooth muscle during aging. Circ. Res. 88, 210-234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D'Ocon, P., Vila, E., 2002. Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832-840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 microM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2+ ATPase by thapsigargin (TG, 1 microM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2+ from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 microM), caused a greater increase in intracellular Ca2+ and tension in aged rats, indicating that mitochondria may accumulate more Ca2+ during aging. The present results show that changes in intracellular Ca2+ stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2+ homeostasis and cell damage.

Mitochondrial alterations and apoptosis in smooth muscle from aged rats.

We studied changes in mitochondrial morphology and function in the smooth muscle of rat colon. Under confocal microscopy, tissues loaded with potentiometric dye displayed rapid and spontaneous depolarization. Cyclosporin A (CsA), inhibitor of the permeability transition pore (PTP), caused an increase in mitochondrial membrane potential (DeltaPsim) in tissues from adult young animals. In aged rats these changes were not observed. This suggests that physiological activation of PTP in aged rats is reduced. Electron microscopy showed alterations of the mitochondrial ultrastructure in tissues from aged rats involving a decreased definition of the cristae and fragmentation of the mitochondrial membranes. We also detected an increase in apoptotic cells in the smooth muscle from aged animals. Our results show that the aging process changes PTP activity, the ability to maintain DeltaPsim and mitochondrial morphology. It is suggested that these can be associated with mitochondrial damage and cell death.

Up-regulation of Ca(2+) channels in vas deferens after chronic treatment of newborn rats with nifedipine.

Radioligand binding and contraction techniques were used to verify if L-type Ca(2+) channels are modified in rat vas deferens after treatment with the blocker nifedipine (15 microg), injected at 7, 14, 21 and 28 days after birth. Vas deferens tissue was used 10, 30 and 90 days after the last injection, to verify if modifications are persistent. Binding studies with cell membranes, using [(3)H]isradipine, showed an increase of the density (B(max)) of Ca(2+) channels by more than 60%, after 10 and 30 days, without changes of affinity (K(d)). Maximal contractions (E(max)) of KCl, were increased by 106% and 37%, respectively, after 10 and 30 days, without changes of apparent affinity (pD(2)). After 90 days, the values of B(max), K(d), E(max) and pD(2) were not different from the controls. Differences were also not found for rats injected when adult. It is concluded that treatment of newborn, but not of adult, rats with nifedipine produced a long-lasting, though reversible, up-regulation of L-type Ca(2+) channels.

Nantenine blocks muscle contraction and Ca2+ transient induced by noradrenaline and K+ in rat vas deferens.

The effect of nantenine, an aporphine alkaloid isolated from Ocotea macrophylla H.B.K., was studied on contractions and Ca(2+) translocation induced by noradrenaline, Ca(2+), or K(+) in the isolated rat vas deferens from reserpinized animals. Concentration-response curves of calcium chloride (CaCl(2)) were performed in the vas deferens, in a Ca(2+)-free nutrient solution, using potassium chloride (KCl, 80 mM) as a depolarizing agent. In these conditions, nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly reduced the maximum contractions (E(max)) of Ca(2+) (IC(50)=2.6 x 10(-4) M) and noradrenaline (IC(50)=2.9 x 10(-4) M). The contractile responses were totally recovered after the withdrawal of nantenine. In addition, experiments performed to measure simultaneously the contraction and the increase of intracellular Ca(2+) induced by noradrenaline (10(-5) M) or KCl (80 mM) showed that nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly decreased both effects. The results suggest that a reversible block of Ca(2+) entry could be involved on the non-competitive-like antagonism of nantenine in rat vas deferens.

Contractile responses of the rat vas deferens after epithelium removal.

The purpose of the present investigation was to verify the role of the epithelium in the functional response of the rat vas deferens. Our results showed that the contractile effect of cumulative doses of clonidine (3.10(-5)-3.10(-3)) was increased after the removal of the epithelium. The effect of clonidine in epithelium-free vas deferens returned to normal values when an isolated epithelium from another vas deferens was added to the organ bath, showing that the epithelium is responsible for this increase of maximum effect for clonidine. Drugs functionally or structurally related to clonidine, such as oxymetazoline, alpha-methylnorepinephrine and moxonidine, did not have their dose-response curves altered. The curves for other contractile agents, such as noradrenaline, acetylcholine, ATP, 5HT, bradykinin and histamine, or the relaxation induced by isoprenaline and forskolin were also not modified. Electrically-induced contractions at frequencies from 0.1 to 20 Hz and the mechanism of negative feed-back, brought about by clonidine (10(-10)-10(-8) M) through pre-synaptic alpha2-adrenoceptors, were not changed after the removal of epithelium. In conclusion, a significant function of the epithelium in the contractility of the rat vas deferens was demonstrated for clonidine, but not for other agonists.

A comparison of histamine effects on the sympathetic neurotransmission of testicular capsule and rat vas deferens.

Histamine is an important modulatory agent of the sympathetic neurotransmission, but its exact action on the testicular capsule or rat vas deferens is not fully understood. The present study sought to further investigate the functional effects of histamine on the neuronal and exogenous noradrenaline-induced contraction of the testicular capsule and rat vas deferens as well as to evaluate the contractile properties of this drug. The testicular capsule or vas deferens from Wistar rats, 3-4 months old, weighing 300-400 g, was isolated and mounted in organ baths for functional experiments. The results indicated that the neuronally evoked contraction of the testicular capsule was affected by histamine (10(-10) to 10(-8) M) with participation of inhibitory (H3 receptors) and excitatory (H1 receptors) receptors. Histamine (10(-7) to 10(-4) M) modulated the field-stimulated vas deferens by excitatory (H2 receptors) and inhibitory (H1 receptors) receptors. Histamine was able to decrease the tonic response for noradrenaline-induced contractions with participation of H1 receptors (testicular capsule) and H3 receptors (vas deferens) followed by nitric oxide generation. At high concentration, histamine exerts contractile effects in both tissues. In the testicular capsule, the histamine-induced contractions were related to H1 receptor activation followed by release of prostaglandins. In contrast, the contractile effects of histamine in the vas deferens were related to H2 receptor activation followed by release of catecholamines from sympathetic nerve endings. Therefore, our results indicate that histamine induced several effects on the sympathetic neurotransmission of rat testicular capsule and vas deferens. These effects are dependent on the concentration used and with participation of multiple histamine receptors.

Effects of clonidine in the isolated rat testicular capsule.

The testicular capsule contracts in response to noradrenaline and adrenaline, but the effects of adrenoceptor agonists, as for instance clonidine, had not yet been thoroughly evaluated. The testicular capsule from adult male Wistar rats was isolated and mounted in organ bath and cumulative concentration curves were performed for clonidine and other adrenergic agonists in the absence or presence of α-adrenoceptors antagonists. The order of potency for agonists (pD2) was clonidine=adrenaline>UK 14,304>noradrenaline>phenylephrine>methoxamine. The consecutive curves for clonidine showed desensitization with 3-fold rightward shift and Emax reduction of 40%. The noradrenaline curves were 4.5, 19 and 190-fold less potent after clonidine pretreatment at 10−5, 10−4 or 10−3 M for 10 min, respectively, added to Emax decrease by about 20%. Clonidine (10−5 M for 10 min) was unable to alter the noradrenaline curves if the treatment was made in the presence of idazoxan (α2-adrenoceptor antagonist) whereas prazosin (α1-adrenoceptor antagonist) was ineffective. The effect of idazoxan 3×10−7 M on noradrenaline curves was decreased by 50% after clonidine pretreatment, as reflected by the concentration ratio of 5.2±1.2 (treated tissue) and 10.1±1.0 (untreated tissue). However, the concentration ratio for prazosin 3×10−8 M was unchanged. After phenoxybenzamine (irreversible antagonist of α1-adrenoceptor) pretreatment, the residual noradrenaline contraction was antagonized by idazoxan or prazosin with pKB values of 7.8 and 5.1, respectively. The results indicate the presence of α2-adrenoceptors in testicular capsule. Furthermore, these receptors may be desensitized by clonidine, causing a decreased potency of noradrenaline.

Increase of angiotensin-converting enzyme activity and peripheral sympathetic dysfunction could contribute to hypertension development in streptozotocin-induced diabetic rats.

Diabetes augments the risk of hypertension. Although several factors have been implicated in the development of such hypertensive state, we designed this study to investigate blood pressure development, the activity of angiotensin-converting enzyme (ACE) in blood as well as sympathetic neurotransmission in the vas deferens of diabetic rats. We used streptozotocin (STZ)-induced diabetic rats (60 mg/kg) in order to evaluate the systolic blood pressure (SBP), ACE activity and peripheral sympathetic neurotransmission. We observed the following changes of parameters: increase of SBP, decrease of heart rate, augmentation of plasma ACE activity, enhancement of phasic and tonic vas deferens contractions elicited by electrical stimulation at 5 Hz, increase of maximal response to noradrenaline (NA) and decrease of adenosine triphosphate (ATP)-elicited contraction of vasa deferentia. The results reveal that in the development of hypertension in diabetic rats, augmentation of circulating ACE activity precedes the sympathetic dysfunction. Additionally, it seems that the purinergic and noradrenergic neurotransmission is compromised.

Functionomics: the analysis of a postgenomic concept on the basis of pregenomic pharmacological studies in smooth muscle

The term functionomics (Amin 2003, Neumann et al. 2004) refers to a postgenomic integrated Systems Biology (Attur et al. 2002) using a multidimensional approach for cells, tissues and organs. It considers current or future involvement among genomics, proteomics or metabolomics, including the main factors that cause biological responses and modulation under different conditions. Our objective in the present review is to summarize the contemporary understanding of functionomics of smooth muscle pharmacology, based on the results obtained on the pregenomic era during several years in our laboratory. The present approach is based on the knowledge of the dynamics of the receptor system, which comprises a cascade of phenomena, leading from the drug administration to the final biological response. We will describe several conditions in which the final effect is modified, based on perturbations induced on drug absorption, distribution, metabolism, interaction with receptors and mobilization of second messengers, as well as by interactions with a second receptor system. We will also discuss the gaps that need to be fulfilled in order to obtain a clear and better understanding of the receptor system in smooth muscle, and to narrow the bridge between ourknowledge of the function of biological systems, genomics, and other recently introduced areas.

O termo funcionômica (Amin 2003, Neumann et al. 2004) refere-se a um estudo posgenômico de Biologia de Sistemas (Attur et al. 2002), usando um enfoque multidimensional, dinâmico e simultâneo para células, tecidos e órgãos. Considera o envolvimento presente e futuro da genômica, proteômica e metabolômica incluindo os principais fatores que causam a resposta biológica final e sua modulação em diferentes condições. Nosso objetivo na presente revisão é resumir o nosso conhecimento atual em relação à funcionômica da farmacologia da musculatura lisa, baseada em resultados que obtivemos ainda na era pregenômica, durante vários anos em nosso laboratório. O presente enfoque baseia-se no que sabemos hoje em dia sobre a dinâmica do sistema receptor, que compreende uma cascata de fenômenos, que vão desde a administração de uma droga até a resposta biológica. Descreveremos várias condições nas quais a resposta é modificada, com base em perturbações produzidas na absorção, distribuição e metabolismo de fármacos, interação com receptores, mobilização de segundos mensageiros, bem como interações com um segundo sistema receptor. Discutiremos também o papel da genômica e as inúmeras falhas que devem ser preenchidas, para que se chegue a um conhecimento integrado e cada vez melhor dos sistemas receptores na musculatura lisa e para encurtar a ponte entre as funções do sistema biológico, genômica e outras áreas recentemente introduzidas.

Voltage-gated potassium currents in rat vas deferens smooth muscle cells.

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.