Adrenergic Terminals Research Articles

6-Nitrodopamine potentiates contractions of rat isolated vas deferens induced by noradrenaline, adrenaline, dopamine and electric field stimulation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that is released from the rat isolated vas deferens, and has been characterized as a major modulator of the contractility of rat isolated epididymal vas deferens (RIEVD). Drugs such as tricyclic antidepressants, α1 and β1β2 adrenoceptor blockers, act as selective antagonists of the 6-ND receptor in the RIEVD. In the rat isolated atria, 6-ND has a potent positive chronotropic action and causes remarkable potentiation of the positive chronotropic effects induced by dopamine, noradrenaline, and adrenaline. Here, whether 6-ND interacts with the classical catecholamines in the rat isolated vas deferens was investigated. Incubation with 6-ND (0.1 and 1 nM; 30min) caused no contractions in the RIEVD but provoked significant leftward shifts in the concentration-response curves to noradrenaline, adrenaline, and dopamine. Pre-incubation of the RIEVD with 6-ND (1 nM), potentiated the contractions induced by electric-field stimulation (EFS), whereas pre-incubation with 1 nM of dopamine, noradrenaline or adrenaline, did not affect EFS-induced contractions. In tetrodotoxin (1 μM) pre-treated (30 min) RIEVD, pre-incubation with 6-ND (0.1 nM) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline, adrenaline, or dopamine. Pre-incubation of the RIEVD with the α2A-adrenoceptor antagonist idazoxan (30 min, 10 nM) did not affect dopamine, noradrenaline, adrenaline, and EFS-induced contractions. However, when idazoxan (10 nM) and 6-ND (0.1 nM) were simultaneously pre-incubated (30 min), a significant potentiation of the EFS-induced contractions of the RIEVD was observed. 6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline, and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, possibly via pre-synaptic adrenoceptors.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

Acute Effect of Selective Chemical Inactivation of Sympathetic or Parasympathetic Atrial Ganglionated Plexus Structures on Atrial Fibrillation Inducibility in Pigs.

We studied the role of both parts of the autonomic intracardiac nervous system in the pathogenesis of atrial fibrillation (AF). In 12 pigs weighing 39±3 kg, AF was induced by burst stimulation. Chemical inactivation of intrinsic cardiac neurons within the right atria was performed by transendocardial injections of liposomal neuromodulators into the dorsal part of the right atrial wall. Sympathetic and parasympathetic terminals were inactivated with 6-hydroxydopamine (6-OHDA, n=6) and ethylcholine aziridinium ion (AF64A, n=6), respectively. Neuromodulators were encapsulated in liposomes (LS) with diameters of 310±50 nm for OHDA and 290±50 nm for AF64A. LS-6-OHDA and LS-AF64A were injected into the ganglionated plexuses after measuring the baseline effective refractory period and assessing myocardial resistance to AF. These measurements were repeated 90 min after the injections. The optimal doses were 0.2 mg/kg for LS-6-OHDA and 0.4 mg/kg for LS-AF64A (in 4 ml of suspension). Immediately after injections of liposomal neuromodulators, almost all pigs showed an increase in HR, and a short-term BP elevation was observed in the LS-AF64A group. At the end of the experiment, similar decrease in the effective refractory period and similar increase in the resistance to AF were observed in all animals. Thus, selective chemical inactivation of cholinergic and adrenergic terminals of the intracardiac nervous system with liposomal neuromodulators increased the resistance to AF in an acute experiment. However, the short observation period does not allow making a definite conclusion about the role of the autonomic nervous system in the pathogenesis of AF, which requires verification of the obtained data in a chronic experiment.

Tetrodotoxin-insensitive electrical field stimulation-induced contractions on Crotalus durissus terrificus corpus cavernosum.

Reptiles are the first amniotes to develop an intromitent penis, however until now the mechanisms involved in the electrical field stimulation-induced contraction on corpora cavernosa isolated from Crotalus durissus terrificus were not investigated. Crotalus and rabbit corpora cavernosa were mounted in 10 mL organ baths for isometric tension recording. Electrical field stimulation (EFS)-induced contractions were performed in presence/absence of phentolamine (10 μM), guanethidine (30 μM), tetrodotoxin (1 μM and 1mM), A-803467 (10 μM), 3-iodo-L-Tyrosine (1 mM), salsolinol (3 μM) and a modified Krebs solution (equimolar substitution of NaCl by N-methyl–D-glucamine). Immuno-histochemistry for tyrosine hydroxylase was also performed. Electrical field stimulation (EFS; 8 Hz and 16 Hz) caused contractions in both Crotalus and rabbit corpora cavernosa. The contractions were abolished by previous incubation with either phentolamine or guanethidine. Tetrodotoxin (1 μM) also abolished the EFS-induced contractions of rabbit CC, but did not affect EFS-induced contractions of Crotalus CC. Addition of A-803467 (10 μM) did not change the EFS-induced contractions of Crotalus CC but abolished rabbit CC contractions. 3-iodo-L-Tyrosine and salsolinol had no effect on EFS-induced contractions of Crotalus CC and Rabbit CC. Replacement of NaCl by N- Methyl-D-glucamine (NMDG) abolished EFS-induced contractions of rabbit CC, but did not affect Crotalus CC. The presence of tyrosine hydroxylase was identified in endothelial cells only of Crotalus CC. Since the EFS-induced contractions of Crotalus CC is dependent on catecholamine release, insensitive to TTX, insensitive to A803467 and to NaCl replacement, it indicates that the source of cathecolamine is unlikely to be from adrenergic terminals. The finding that tyrosine hydroxylase is present in endothelial cells suggests that these cells can modulate Crotalus CC tone.

Dual allosteric modulation of opioid antinociceptive potency by α2A-adrenoceptors

Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using the hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10–70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2A-KO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management.

Regional innervation of the heart in the goldfish, Carassius auratus: A confocal microscopy study

The intracardiac nervous system represents the final common pathway for autonomic control of the vertebrate heart in maintaining cardiovascular homeostasis. In teleost fishes, details of the organization of this system are not well understood. Here we investigated innervation patterns in the heart of the goldfish, a species representative of a large group of cyprinids. We used antibodies against the neuronal markers zn-12, acetylated tubulin, and human neuronal protein C/D, as well as choline acetyltransferase, tyrosine hydroxylase, nitric oxide synthetase, and vasoactive intestinal polypeptide (VIP) to detect neural elements and their transmitter contents in wholemounts and sections of cardiac tissue. All chambers of the heart were innervated by choline acetyltransferase-positive axons, implying cholinergic regulation; and by tyrosine hydroxylase-containing axons, implying adrenergic regulation. The mean total number of intracardiac neurons was 713 ± 78 (SE), nearly half of which were cholinergic. Neuronal somata were mainly located in a ganglionated plexus around the sinoatrial valves. Somata were contacted by cholinergic, adrenergic, nitrergic, and VIP-positive terminals. Putative pacemaker cells, identified by immunoreactivity for hyperpolarization activated, cyclic nucleotide-gated channel 4, were located in the base of the sinoatrial valves, and this region was densely innervated by cholinergic and adrenergic terminals. We have shown that the goldfish heart possesses the necessary neuroanatomical substrate for fine, region-by-region autonomic control of the myocardial effectors that are involved in determining cardiac output.

Chromogranin A: a novel factor acting at the cross road between the neuroendocrine and the cardiovascular systems

Chromogranin A (CHGA) is a secretory protein stored in and released from neurons and cells of the diffuse neuroendocrine system. Cells of the adrenal medulla and adrenergic terminals are a main source of CHGA but also myocardial cells produce it under stress conditions. After secretion, CHGA is cleaved into several biologically active fragments, including vasostatins and catestatin. CHGA and its proteolytic peptides exert a broad spectrum of activities on the cardiovascular system. They act on blood pressure by controlling the vascular tone and the cardiac inotropic and chronotropic function. CHGA revealed to be a sensitive marker of myocardial dysfunction, with a high predictive power of morbidity and mortality in heart failure and ischemic heart disease. In addition, CHGA has been involved in the control of sustained endothelial inflammation and has been shown to be a good marker of persistent vascular inflammation in rheumatologic disorders affecting vessels.

Control of bone remodeling by nervous system. Regulation of bone metabolism by peripheral nervous system

Bone is richly innervated with adrenergic and peptidergic nerve terminals, and these play important roles in bone remodeling. Recent study confirmed a functional nerve-osteoblastic interplay by using an in vitro co-culture model comprising mouse osteoblastic cells and neurite-spouting mouse superior cervical ganglia. This indicates that nerve-osteoblastic cell cross-talk can occur for the dynamic regulation of local bone metabolism by peripheral nervous system. This review summarized both in vivo and in vitro evidences implicating peripheral neuron action in bone metabolism.

Catestatin Improves Post-Ischemic Left Ventricular Function and Decreases Ischemia/Reperfusion Injury in Heart

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of beta2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by beta2- and beta3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

The involvement of heating rate and vasoconstrictor nerves in the cutaneous vasodilator response to skin warming

Slow local skin heating (LH) causes vasodilator responses, some of which are dependent on sympathetic nerve function. It is not known, however, how the rate of LH affects either the sympathetic or the nonadrenergic components of the responses to LH and whether the adrenergic effects of LH depend on tonic sympathetic activity or whether LH stimulates transmitter release. In part 1, cutaneous vascular conductance (CVC) responses to slow and fast LH (+0.1 degrees and +2 degrees C/min) from 34 degrees to 40 degrees C were compared both at control sites and at sites pretreated with bretylium tosylate (BT; blocks transmitter release from adrenergic terminals). We confirmed, as previously found, the axon reflex (AR) response to slow LH to be blocked by BT (P < 0.05). Pretreatment with BT reduced the AR only with fast LH. BT inhibited the peak vasodilation achieved with both rates of LH (P < 0.05). Longer-term LH was associated with a slow fall in CVC, the classical "die away" phenomenon, at untreated sites (P < 0.05) but not at BT-pretreated sites. Thus the LH-stimulated AR is only partially dependent on intact sympathetic function, and the "die away" phenomenon is dependent on such function. In part 2, we tested whether the conditions in part 1 (whole body and local skin temperatures of 34 degrees C) completely suppressed sympathetic nerve activity. The infusion of BT by microdialysis did not change the CVC (P > 0.05), suggesting the absence of tonic activity in those conditions and therefore that the adrenergic components of the responses in part 1 are via the stimulation of the transmitter release by LH.

Influence of hyperoxia on skin vasomotor control in normothermic and heat-stressed humans

Hyperoxia induces skin vasoconstriction in humans, but the mechanism is still unclear. In the present study we examined whether the vasoconstrictor response to hyperoxia is through activated adrenergic function (protocol 1) or through inhibitory effects on nitric oxide synthase (NOS) and/or cyclooxygenase (COX) (protocol 2). We also tested whether any such vasoconstrictor effect is altered by body heating. In protocol 1 (n = 11 male subjects), release of norepinephrine from adrenergic terminals in the forearm skin was blocked locally by iontophoresis of bretylium (BT). In protocol 2, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) and the nonselective COX antagonist ketorolac (Keto) were separately administered by intradermal microdialysis in 11 male subjects. In the two protocols, subjects breathed 21% (room air) or 100% O(2) in both normothermia and hyperthermia. Skin blood flow (SkBF) was monitored by laser-Doppler flowmetry. Cutaneous vascular conductance (CVC) was calculated as the ratio of SkBF to blood pressure measured by Finapres. In protocol 1, breathing 100% O(2) decreased (P < 0.05) CVC at the BT-treated and at untreated sites from the levels of CVC during 21% O(2) breathing both in normothermia and hyperthermia. In protocol 2, the administration of l-NAME inhibited (P < 0.05) the reduction of CVC during 100% O(2) breathing in both thermal conditions. The administration of Keto inhibited (P < 0.05) the reduction of CVC during 100% O(2) breathing in hyperthermia but not in normothermia. These results suggest that skin vasoconstriction with hyperoxia is partly due to the decreased activity of functional NOS in normothermia and hyperthermia. We found no significant role for adrenergic mechanisms in hyperoxic vasoconstriction. Decreased production of vasodilator prostaglandins may play a role in hyperoxia-induced cutaneous vasoconstriction in heat-stressed humans.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine β-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen’s cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.

Pressor Responses to Ephedrine Are Mediated by a Direct Mechanism in the Rat

The mechanism of the pressor response to ephedrine is controversial. In the present study. i.v. injections of ephedrine increased systemic and pulmonary arterial pressure, and i.a. injections decreased hindlimb blood flow in a dose-related manner. Responses to ephedrine were inhibited by alpha-receptor blocking agents and were not attenuated by blockade of the norepinephrine reuptake transporter (NET) or by catecholamine depletion using reserpine or a combination of reserpine and alpha-methyl-p-tyrosine, whereas responses to tyramine and amphetamine were inhibited by these treatments. The magnitude of the pressor response to ephedrine was similar in anesthetized and conscious rats. Tachyphylaxis developed to pressor responses to ephedrine and amphetamine with sequential injections; however, ephedrine tachyphylaxis differed in that subsequent responses to alpha-receptor agonists were attenuated. These results suggest that the systemic and pulmonary pressor and hindlimb vasoconstrictor responses to ephedrine are mediated by direct action on alpha-adrenergic receptors and that the release of norepinephrine from adrenergic terminals plays no significant role. These results provide support for the hypothesis that responses to ephedrine are directly mediated in the intact rat, whereas responses to amphetamine are mediated in a large part by the release of norepinephrine from adrenergic terminals.

Quantification and pharmacological characterization of dialysate levels of noradrenaline in the striatum of freely-moving rats: release from adrenergic terminals and modulation by α 2-autoreceptors

Information concerning striatal levels of noradrenaline (NA) remains inconsistent. Here we have addressed this issue using a sensitive method of HPLC coupled to amperometric detection. The NA reuptake-inhibitor, reboxetine, selectively elevated levels of NA versus dopamine (DA), and NA levels were also selectively elevated by the α 2-adrenoceptor (AR) antagonist, atipamezole. The actions of atipamezole were mimicked by the preferential α 2A-AR antagonist, BRL44408, while JO-1 and prazosin, preferential antagonists at α 2C-ARs, caused less marked elevations in NA levels. In contrast to antagonists, the α 2-AR agonist, S18616, decreased NA levels and likewise suppressed those of DA. Unilateral lesions of the substantia nigra with 6-hydroxydopamine depleted DA levels without affecting those of NA. Further, the D 3/D 2 receptor agonist, quinelorane, decreased levels of DA without modifying those of NA. However, the D 3/D 2 receptor antagonists, haloperidol and raclopride, and the DA reuptake-inhibitor, GBR12935, elevated levels of both DA and NA. Levels of 5-HT (but not of NA or DA) were increased only by the 5-HT reuptake-inhibitor, citalopram. They were decreased by S18616 and prazosin, reflecting the inhibitory and excitatory influence of α 2- and α 1-ARs, respectively, upon serotonergic pathways. In conclusion, NA in the striatum is derived from adrenergic terminals. Its release is subject to tonic, inhibitory control by α 2-ARs, possibly involving both α 2A- and α 2C-AR subtypes, though their respective contribution requires clarification. A role of dopaminergic terminals in the reuptake of NA likely explains the elevation in its levels elicited by DA reuptake-inhibitors and D 3/D 2 receptor antagonists.

Effect of acute intravenous cocaine administration on endothelium-dependent vasodepressor responses to acetylcholine.

Cardiovascular events commonly associated with acute and chronic cocaine abuse include coronary vasospasm, arrhythmias, myocardial infarction, and sudden death. It has been suggested that cocaine causes endothelial dysfunction and vasoconstriction by inhibiting local production of nitric oxide and that endothelial dysfunction may be involved in the cardiovascular events associated with cocaine use. The present study investigated the effect of acute intravenous cocaine administration on endothelium-dependent vasodepressor responses to acetylcholine in the anesthetized rat. Rats were anesthetized with intraperitoneally Inactin (140 mg/kg) and catheters were inserted into the jugular vein and iliac artery for the injection of drugs and measurement of systemic arterial pressure. A thermistor catheter was advanced to the aortic arch for the measurement of cardiac output via the thermal dilution technique. A 5 mg/kg intravenous dose of cocaine, which enhances the vasopressor response to norepinephrine and blocks the vasopressor response to tyramine, had no effect on the vasodepressor response to the endothelium-dependent vasodilator acetylcholine. In addition, responses to the nitric oxide-donor sodium nitroprusside and the vasoconstrictor peptide angiotensin II were not altered by administration of cocaine. Acute administration of cocaine in a dose that blocks norepinephrine uptake into adrenergic terminals had no effect on endothelial function as measured by the absence of an effect on the vasodepressor response to intravenous injections of acetylcholine. Although chronic cocaine exposure has been shown to cause vascular dysfunction, eventually leading to impairment of the nitric oxide pathway, it appears that acute cocaine administration does not inhibit this pathway in the anesthetized rat.

Adrenergic control of protein metabolism in skeletal muscle.

This review summarizes evidence indicating that the sympathetic nervous system, through hormonal and neurotransmitter actions, produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed which indicate that catecholamines secreted by the adrenal medulla have an inhibitory effect on muscle Ca(2+)-dependent protein degradation independently of other hormones. In addition, norepinephrine released from adrenergic terminals may increase the rate of protein synthesis in oxidative muscles, leading to increased protein accretion. Evidence is also presented that these effects seem to be mediated by beta(2)-adrenoceptors and cyclic adenosine monophosphate-dependent pathways. The understanding of the precise mechanisms by which endogenous catecholamines promote muscle anabolic effects may bring new perspectives for efficient treatment of muscle-wasting conditions and enhancement of growth efficacy in farm species.

A scanning and transmission electron microscopic study of contractile trabecules in the rat spleen.

The fine structures of contractile trabecules in the splenic red pulp of the rat were examined by electron microscopy to elucidate their participation in the active contraction of the spleen. Numerous fine thready trabecules were developed in the red pulp. They were enveloped with a cytoplasmic layer of reticular cells and consisted of elongated smooth muscle cells, fascicles of collagenous fibrils and elastic fibers. Their fibrous components in the capsular ends extended in a triangular form of fanribs into the fibrous tunica of the capsule. Smooth muscle cell-like interstitial cells (SIC) were situated in the interfibrous spaces. Flattened SICs were affixed with cytoplasmic processes to the elastic lamina. The trabeculocapsular junctions were represented on the elastic lamina by grouped or isolated circular patches with concentrically arranged triangular processes and were also observed on the capsular serosa by plaques with scarce microvilli of serosal cells. Smooth muscle cells of the fine trabecules were equipped on the cell surface with anchoring structures to extracellular fibrous elements as previously described by Gabella (1981). Close associations were also seen between the smooth muscle cells and elastic fibers which were terminated to the fascicles of collagenous fibers. Cell-to-cell connections were expressed by fibrous connections between spiny processes and a small number of puntate intermediate junctions and nexuses. Unmyelinated nerve fibers with adrenergic terminals were seen in the intercellular spaces. We propose that for the rat spleen, the fine trabecules in the red pulp are muscular contractiles which are responsible for the active contraction due to sympathetic stimuli and the administration of alpha 1-adrenoceptor agonists, while the elastic lamina in the capsule plays a role in the comprehensive contraction of the subcapsular vascular bed.

N-methyl- d-aspartate-type glutamate receptors are found in post-synaptic targets of adrenergic terminals in the thoracic spinal cord

Adrenergic (C1) neurons in the rostral ventrolateral medulla (RVL) are sympathoexcitatory and project directly to sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord. C1 neurons also contain glutamate which may mediate the excitatory effects of RVL stimulation on SPNs through the N-methyl- d-aspartate (NMDA)-type receptor. Dual-labeling immunocytochemistry, combined with electron microscopy, was used to determine if NMDA receptors are located post-synaptic to adrenergic terminals in the spinal cord. Adrenergic terminals were labeled using an antibody directed against phenylethanolamine- N-methyl transferase (PNMT) and the NMDA receptor was identified using an antibody directed against the R1 subunit of the receptor (NMDAR1). NMDAR1 was found primarily in large and small dendrites and a few perikarya. The presence of NMDAR1 in the dendritic targets of PNMT-containing terminals was quantified in spinal cords sectioned either horizontally or coronally. PNMT-labeled terminals formed asymmetric synapses on dendrites containing immunogold labeling for NMDAR1, but NMDAR1 was more often detected in the targets of PNMT terminals when spinal cords were sectioned horizontally (59%) rather than coronally (28%). This difference in prevalence of NMDAR1 in targets of PNMT terminals is likely due to the preferential orientation of SPN dendrites in the horizontal plane, since longer dendritic shafts were visible in horizontal sections. When NMDAR1 was present in the dendritic targets of many adrenergic terminals, it was usually located at sites distal to the adrenergic input. We conclude that NMDA receptor ligands are likely to modulate the activity of dendritic targets of adrenergic terminals in the spinal cord, but are not closely associated with adrenergic synaptic input.

Effects of two putative endogenous digitalis-like factors, marinobufagenin and ouabain, on the Na+,K+-pump in human mesenteric arteries

Recently, in rat aortae, two putative digitalis-like factors, marinobufagenin and ouabain, were shown to interact with alpha-1 (sarcolemma) and alpha-3 (nerve endings) subunits of the sodium pump, respectively, and elicit vasoconstriction via inhibition of the Na+,K+-pump in vascular smooth muscle or norepinephrine release. The purpose of the present study was to investigate the effects of ouabain and marinobufagenin on vascular tone, activity of Na+,K+-pump, and the expression of isoforms of the Na+,K+-ATPase alpha-subunit in human mesenteric arteries. Arteries were obtained from male patients undergoing surgery due to intestinal adenocarcinoma. Vasoconstrictor effects of both inhibitors were studied in isolated vascular rings. Na+,K+-pump activity was measured using the 86Rb technique. Membrane fractions of sarcolemma and nerve endings plasmalemma were prepared via differential centrifugation of membranes in a sucrose density gradient Specific antibodies to the alpha-1 and alpha-3 subunits of Na+,K+-ATPase were used to detect alpha-1 and alpha-3 isoforms in the membrane fractions by Western blotting. Marinobufagenin (EC50 = 88+/-15 nmoles/l) and ouabain (EC50 = 320+/-50 nmoles/l) elicited vasoconstriction in mesenteric artery rings. At a concentration of 1 nmol/l, both compounds stimulated the Na+,K+-pump, but inhibited its activity at 10-1000 nmoles/l. No stimulation of the Na+,K+-pump was observed in the presence of 5 micromol/l phentolamine; rather 1 nmol/l of marinobufagenin and ouabain inhibited the Na+,K+-pump by 30% and 13%, respectively. The alpha-1 polyclonal antibody detected alpha-1 isoform in membrane fractions from both sarcolemma and nerve endings. A monoclonal alpha-1 antibody detected the material in sarcolemmal membranes only. The alpha-3 isoform was detected in both membrane fractions by both antibodies, but staining for alpha-3 was more pronounced in the nerve endings. These results demonstrate that, in physiologically 'realistic' concentrations, marinobufagenin and ouabain can significantly affect the Na+,K+-pump in human mesenteric artery, and illustrate the importance of interaction of digitalis-like Na+,K+-pump inhibitors with the Na+,K+-ATPase localized to the intravascular adrenergic terminals. Present observations are in accord with the previous data suggesting that marinobufagenin and ouabain display greater affinity to alpha-1 and alpha-3 isoforms of the Na+,K+-pump, respectively, and support the view that the differential responsiveness to endogenous digitalis-like inhibitors is one of the features of alpha-isoforms of Na+,K+-ATPase.