Heart failure-induced changes of voltage-gated Ca2+ channels and cell excitability in rat cardiac postganglionic neurons.

Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia and sympathetic (CPS) neurons located in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single-cell real-time PCR and immunofluorescent data showed that L (Ca(v)1.2 and Ca(v)1.3), P/Q (Ca(v)2.1), N (Ca(v)2.2), and R (Ca(v)2.3) types of Ca2+ channels were expressed in CPP and CPS neurons, but CHF decreased the mRNA and protein expression of only the N-type Ca2+ channels in CPP neurons, and it did not affect mRNA and protein expression of all Ca2+ channel subtypes in the CPS neurons. Patch-clamp recording confirmed that CHF reduced N-type Ca2+ currents and cell excitability in the CPP neurons and enhanced N-type Ca2+ currents and cell excitability in the CPS neurons. N-type Ca2+ channel blocker (1 µM ω-conotoxin GVIA) lowered Ca2+ currents and cell excitability in the CPP and CPS neurons from sham-operated and CHF rats. These results suggest that CHF reduces the N-type Ca2+ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca2+ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.

Inability to walk, disequilibrium, incoherent speech, disorientation following the instillation of 1% cyclopentolate eyedrops: case report.

A 4-year-old boy, who had no prior history of convulsions, presented with inability to walk, disequilibrium, dysarthria (incoherent speech), and impaired cognition (disorientation) following the instillation of 1% cyclopentolate, a commonly used mydriatic in pediatric practice. This case demonstrates the uncommon, although serious, atropine-like adverse effect of cyclopentolate eyedrops in usual dosage in child.

A novel gel based vehicle for the delivery of acetylcholine in quantitative sudomotor axon reflex testing.

This study describes a novel gel based vehicle for the delivery of acetylcholine (ACh) during quantitative sudomotor axon reflex testing (QSART). A dose and current response study were undertaken on 20 healthy control participants to characterize the efficiency of a gel based vehicle for the delivery of ACh. Values obtained for total sweat volume and latency to sweat onset with gel iontophoresis of ACh during QSART were comparable to previously published normative data using solution based vehicles. Patient discomfort, utilizing the gel based vehicle during the QSART procedure, was minimal. Improvement in iontophoresis using the gel formulation as a vehicle for ACh delivery has the potential to lower the voltage required to overcome skin resistance during QSART and may result in improved patient comfort during the procedure.

Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic ganglia and bladder.

PURPOSE: We examined neurotrophin and receptor tyrosine kinase (Trk) expression in the bladder and major pelvic ganglia (MPG) after cyclophosphamide induced cystitis in rats. MATERIALS AND METHODS: The bladder and MPG were used in immunohistochemical studies, enzyme-linked immunoassays and Western blots for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), TrkA and TrkB. Bladder postganglionic MPG cells were labeled by tracing techniques. RESULTS: NGF and BDNF expression was decreased in the bladder of all rats after cystitis (p < or =0.001). NGF and BDNF expression was increased in the MPG in male rats with cystitis (p < or =0.01). Cells expressing TrkA and TrkB immunoreactivity (IR) increased 78% to 81% in the MPG in male rats with cystitis. TrkA-IR or TrkB-IR bladder postganglionic cells increased 50% to 74% with cystitis. Cystitis increased TrkA-IR 5 to 10-fold and TrkB-IR 10 to 12-fold in detrusor muscle. TrkA-IR and TrkB-IR were prominent in control urothelium but decreased with cystitis. After cystitis TrkB-IR nerve fibers and TrkA-IR cellular infiltrates were more apparent compared to controls. CONCLUSIONS: Cystitis decreases bladder NGF and BDNF expression, whereas MPG expression is increased. This change may reflect neurotrophin release at the bladder and retrograde transport to the MPG. TrkA-IR and TrkB-IR are increased in bladder postganglionic cells and bladders with cystitis. This increase may reflect a shift in Trk staining from urothelium to detrusor muscle and nerve fibers with cystitis. Neurotrophin/Trk interactions in the bladder and MPG may contribute to bladder overactivity with cystitis.

Androgen induced norepinephrine release from postganglionic neurons mediates accessory sex organ smooth muscle proliferation.

PURPOSE: Guinea pig seminal vesicle smooth muscle displays an initial androgen dependent, proliferative response during early puberty, followed by progression to an androgen resistant, amitotic state in adults. We determined the role of norepinephrine in androgen dependent pubertal proliferation and in the subsequent terminal differentiation of adult seminal vesicle smooth muscle. MATERIALS AND METHODS: Guinea pig seminal vesicle provided a suitable model since its unique anatomy allowed clean harvest of smooth muscle without epithelium. Norepinephrine release from postganglionic adrenergic nerve terminals in seminal vesicle smooth muscle was measured using several techniques. Prazosin sensitive electrical field stimulation of contractile responses qualitatively assessed norepinephrine release. Norepinephrine release was quantified directly in vitro from incubated seminal vesicle smooth muscle minces and indirectly ex vivo from intact tissue using the endogenous seminal vesicle smooth muscle concentration ratio of 3,4-dihydroxyphenylglycol-to-norepinephrine (Sigma Chemical Co., St. Louis, Missouri). Norepinephrine mediated seminal vesicle smooth muscle proliferation was assessed by the time course relationships of androgen induced norepinephrine release, protein kinase C activation-depletion and increases in total DNA, the impact of in vivo reserpine induced norepinephrine depletion on protein kinase C activation-depletion and the mitogenic response, and the alpha1-adrenoceptor mediated mitogenic response in cultured seminal vesicle smooth muscle cells. RESULTS: In prepubertal smooth muscle androgen induced norepinephrine release from postganglionic neurons. The effect was independent of preganglionic innervation. Increased norepinephrine release was concurrent with the onset of androgen induced protein kinase C activation-depletion and cellular proliferation. In vivo norepinephrine depletion to 1% or less of control values by chronic reserpine treatment selectively antagonized the androgen induced increases in smooth muscle DNA and protein kinase C down-regulation. Norepinephrine depletion by reserpine neither induced apoptosis nor altered cell number. Cell culture experiments demonstrated that alpha1-adrenoceptors mediated the proliferative response to norepinephrine. Together these findings indicate that increased norepinephrine release has an obligatory role in androgen dependent muscle cell proliferation during puberty. Terminally differentiated smooth muscle in adults was characterized by androgen resistance to elevated norepinephrine release and protein kinase C activation. CONCLUSIONS: Androgen induced norepinephrine release from postganglionic neurons in seminal vesicle smooth muscle mediated the proliferative response that occurs in early pubertal development. Normal uncoupling of elevated norepinephrine release and protein kinase C activation-depletion may represent a key event in the normal terminal differentiation of accessory sex organ smooth muscle in adults.

A possible indirect sympathomimetic action of metformin in the arterial vessel wall of spontanously hypertensive rats.

The antidiabetic drug metformin (MF) typically achieves only micromolar levels in plasma with normal therapeutic use. However, it is also known to accumulate in various tissues up to several times higher after standard oral dosing and we now have evidence from both in vivo and in vitro experiments with spontaneously hypertensive rats (SHR) that millimolar levels stimulate release of norepinephrine (NE) from vascular sympathetic nerve endings (SNEs). As shown in the present work with SHR tail arterial tissue (rich in SNEs), the known vasodilator effect of millimolar levels of MF on the smooth muscle (even if contracted with a nonadrenergic agonist), is attenuated by the presence of the SNEs unless phentolamine (an alpha receptor blocker) is present. We reasoned that the mechanism for this apparent NE-releasing action of MF is not exocytotic release as that would require depolarization of the neuronal cell membranes in SNEs, and MF at millimolar levels is known to repolarize (not depolarize) membranes of other cells. Thus, we tested the possibility that MF releases NE by an indirect sympathomimetic-like action. Such an action should be amplified by monoamine oxidase inhibitors (e.g. iproniazid) and blocked by NE-carrier inhibitors (e.g. desipramine). Accordingly, we found that the abovementioned attenuating effect of intact SNEs on MF's relaxation of SHR tail arterial tissue (compared to tissues in which SNEs were experimentally removed with 6-hydroxydopamine) was amplified nearly 3-fold by iproniazid (p<0.05) and blocked by desipramine (p<0.05). These results support an indirect sympathomimetic action of MF and raise the question whether commonly used antidepressants with properties similar to iproniazid and desipramine might alter MF's beneficial vasodilatory (and thus antihypertensive) effectiveness in diabetic patients with hypertension.

Substance P evokes bradycardia by stimulation of postganglionic cholinergic neurons.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 microl saline) caused a brief decrease in heart rate (-30+/-3 beats/min from a baseline of 256+/-4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28+/-2 mmHg from baseline of 51+/-5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 +/- 9 beats/min before vs -8 +/- 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 +/- 6 beats/min before versus -74 +/- 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons.

A study on possible modulating and direct effects of gamma2-MSH and ACTH-(1-24) on the cardiovascular system of the rat.

In conscious rats, gamma2-melanocyte-stimulating hormone (gamma2-MSH) dose-dependently increases blood pressure and heart rate, whereas adrenocorticotropin-(1-24) [ACTH-(1-24)] dose-dependently decreases blood pressure, an effect which was accompanied by a reflectory tachycardia. As the exact mechanism involved in these cardiovascular effects of the two melanocortins is as yet not known, we undertook a series of experiments to investigate the possibility that these peptides have modulating or direct effect on the cardiovascular system of the rat. In pithed rats gamma2-MSH, administered intravenously (i.v.) in doses of 5-200 nmol/kg, had no significant effect on systolic and diastolic blood pressure and on heart rate, whereas ACTH-(1-24), 5-500 nmol/kg, i.v., dose-dependently decreased blood pressure and increased heart rate. Infusion of gamma2-MSH, 10(-8) M, or ACTH-(1-24), 10(-6) M, in the isolated perfused rat heart did not significantly affect left ventricular pressure or coronary flow. Pretreatment with either gamma2-MSH or ACTH-(1-24) did not modify the responsiveness of the myocardium and coronary vasculature to salbutamol and phenylephrine. Neither gamma2-MSH nor ACTH-(1-24) did affect the vascular contractile machinery of skinned vascular smooth muscles of the rabbit with respect to Ca2+ handling in the cell, as measured by its sensitivity to exogenously applied Ca2+. Gamma2-MSH had no effect on blood pressure and heart rate in pithed rats in which postganglionic sympathetic outflow was stimulated by 1,1-dimethyl-4-phenylpiperazinium (DMPP), nor in pithed rats in which preganglionic sympathetic outflow was stimulated electrically. A dose of 15 nmol/kg ACTH-(1-24) had no significant influence on preganglionic outflow to the cardiac and vascular structures in pithed rats. These data show that gamma2-MSH does not exert its cardiovascular effects via a peripheral site of action at the level of the vascular system and the heart, nor directly on pre- or postganglionic sympathetic outflow. These results are in support for the notion that the peptide acts via a brain region localised outside the blood-brain barrier. The acute depressor effect of ACTH-(1-24), however, seems to be due to a direct effect on the vasculature in the periphery.

Responses of rat postganglionic sympathetic vasoconstrictor neurons following blockade of nitric oxide synthesis in vivo.

The hypothesis was tested that the activation of postganglionic sympathetic neurons contributes to the peripheral vasoconstriction and the blood pressure increase which are observed in rats after systemic blockade of nitric oxide synthase by substituted L-arginine analogues. Single and multifiber postganglionic sympathetic activity supplying hindlimb hairy skin and the activity in the caudal lumbar sympathetic trunk supplying mainly hindlimb skeletal muscle were recorded in anaesthetized, paralysed and artificially ventilated Wistar rats before, during and up to 1 h after intravenous injection of a supramaximal dose (10 or 35 mg/kg) of N(G)-nitro-L-arginine methyl ester. This elicited a sustained rise of arterial blood pressure, a long-lasting decrease in heart rate and vasoconstriction in hindlimb skin and skeletal muscle as measured by laser Doppler flowmetry. With intact buffer nerves all sympathetic neurons analysed responded with a decrease in their ongoing activity in parallel with the vasoconstriction and the increased blood pressure, except for one neuron which was unresponsive. These responses were probably mediated by the arterial baroreceptors. since it was shown that N(G)-nitro-L-arginine methyl ester did not impair the function of both the afferent and the efferent limb of the reflex. Furthermore, baroreceptor denervation almost abolished the inhibitory responses in sympathetic neurons. In baroreceptor denervated animals, with a latency of about 15 min after N(G)-nitro-L-arginine methyl ester there was an increase in sympathetic activity without a further increase in blood pressure. It was concluded that sympathetic vasoconstrictor neurons which supply the rat hindlimb do not contribute by neurogenic means to the vasoconstriction and the blood pressure increase occurring after blockade of the nitric oxide synthase. Instead, the results suggest that sympathetic vasoconstrictor neurons, via the baroreceptor loop, counteract the vasoconstriction caused by the blockade of endothelium-derived nitric oxide. Therefore nitric oxide does not seem to play a role in the central regulation of activity in the sympathetic vasoconstrictor pathways studied here. The long latency increase in sympathetic activity observed after N(G)-nitro-L-arginine methyl ester in baroreceptor-denervated animals may be due to an impairment of blood flow in the brainstem.

Effect of byakko-ka-ninjin-to on the efferent activity of the autonomic nerve fibers innervating the sublingual gland of the rat.

The effect of intraduodenal infusion of Byakko-ka-ninjin-to (BN) on the efferent activity of the autonomic outflow to the sublingual gland was observed in the anesthetized rat. Intraduodenal infusion of BN (50-200 mg/kg) resulted in a dose-related increase in efferent activity. The enhancement of the nerve activity following administration of 200 mg/kg BN lasted longer than three hours. It was observed that the suppressive effect on efferent activity due to i.v. administration of hypertonic saline was antagonized by intraduodenal infusion of BN. From these observations it is suggested that Byakko-ka-ninjin-to acts as a facilitatory agent on salivary secretion.

Alpha 1-adrenoceptor-mediated sympathetically dependent mechanical hyperalgesia in the rat.

The model of rolipram (a type IV phosphodiesterase inhibitor) induced prolongation (> 3 days) of the mechanical hyperalgesia produced by the intradermal injection of prostaglandin E2 in the hairy skin of the hindpaw of the rat, measured by the Randall-Selitto paw-withdrawal test, was employed to study mechanisms involved in the contribution of the sympathetic postganglionic neuron to mechanical hyperalgesia. Lumbar surgical sympathectomy prevented rolipram-induced prolongation of prostaglandin E2 hyperalgesia. Decentralization of sympathetic postganglionic neurons innervating the hindpaw did not, however, effect rolipram-induced prolongation of prostaglandin E2 hyperalgesia. Phentolamine, an alpha-adrenoceptor antagonist, and prazosin, an alpha 1-selective adrenoceptor antagonist, when given systemically or intradermally at the site of injection of prostaglandin E2 and rolipram, blocked rolipram-induced prolongation of prostaglandin E2 hyperalgesia. Intrathecal administration of phentolamine and prazosin were, however, without effect on rolipram-induced prolongation of prostaglandin E2 hyperalgesia. Yohimbine, an alpha 2-adrenoceptor antagonist given systemically, intradermally or intrathecally also did not produce any alteration in rolipram-induced prolongation of prostaglandin E2 hyperalgesia. We propose that sympathetic postganglionic neurons are involved in rolipram-induced prolongation of prostaglandin E2 hyperalgesia and that this form of sympathetically dependent hyperalgesia, which is independent of activity in preganglionic sympathetic neurons, is mediated by a peripheral alpha 1-adrenergic mechanism.

[Pholedrine for determining the site of Horner syndrome]. / Pholedrin zur Lokalisation des Horner-Syndroms.

BACKGROUND: Pharmacologic testing by indirect acting sympathomimetics like hydroxyamphetamine may determine the site of the lesion in Horner's syndrome. Pholedrine is chemically similar to hydroxyamphetamine. Therefore we examined if it shows the same effects in normal subjects and in patients with Horner's syndrome. METHODS: Pupil diameter was measured by means of standardized photography before and after single and with different intervals repeated administration of pholedrine eye drops in normal subjects. In 18 patients with Horner's syndrome and known hydroxyamphetamine test results, a pholedrine test was carried out analogous to the hydroxyamphetamine test. RESULTS: Pholedrine dilates the normal pupil by 2.2 mm (mean). It acts at the longest 8-10 hours with maximal effect between 20 and 90 minutes. After this period its effect decreases rapidly. It acts independently from age and from baseline pupil diameter. Given repeatedly the second administration reaches the same mydriatic effect as the first only if the interval between both applications is 72 hours or more. This is because it needs some time to refill the noradrenaline stores in the sympathetic neuron innervating the dilator muscle of the pupil. In Horner's syndrome pholedrine dilates the involved pupil only minimally in case of a postganglionic lesion, and in case of a preganglionic lesion it dilates the pupil even slightly more than the normal fellow pupil. It shows the same effect as hydroxyamphetamine. There are only few side effects. CONCLUSION: Pholedrine is a substitute for hydroxyamphetamine to localize the site of the lesion in patients with Horner's syndrome.

Nitric oxide modulation of calcium-activated potassium channels in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of calcium-activated potassium channels in cultured neurones of avian ciliary ganglia by sodium nitroprusside and L-arginine. 2. Sodium nitroprusside (100 microM) reduced the net outward current by 22 +/- 1% at 4.8 ms (mean +/- s.e. mean) and 25 +/- 1% at 350 ms during a test depolarization to +40 mV from a holding potential of -40 mV. The outward current remained reduced for the duration of the recording following a single application of sodium nitroprusside. These effects did not occur if the influx of calcium ions was first blocked with Cd2+ (500 microM). Application of ferrocyanide (100 microM) reduced the net outward current by only 6 +/- 3% at 350 ms during a test depolarization to +40 mV. 3. L-Arginine (270 microM) reduced the net outward current on average by 19 +/- 2% at 4.8 ms and 22 +/- 2% at 350 ms during a test depolarization to +40 mV. The current remained in this reduced state for the duration of the recording following a single application of L-arginine. These effects were reduced to 11 +/- 1% at 4.8 ms and 11 +/- 2% at 350 ms in the presence of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. In order to alleviate the dependence of calcium-activated potassium channels (Ik(Ca)) on the inward flux of calcium ions, the patch-clamp pipettes were filled with a solution containing 100 microM CaCl2, and the Ca2+ in the bathing solution was replaced with EGTA. Under these conditions sodium nitroprusside reduced the total outward current during a depolarizing pulse of + 40 mV by 9 +/_ 1% at 4.8 ms and by 36 +/- 3% at 350 ms. L-Arginine (270 microM) reduced this current under the same conditions by 9 +/- 1% at 4.8 ms and by 35 +/- 2% at 350 ms.5. Calcium-activated potassium currents were sensitive to apamin (50 nM), as this reduced the outward current by 23 +/- 3% at 350 ms when a high calcium-containing pipette was used during a depolarizing command to + 40 mV. L-Arginine still decreased the outward current in the presence of apamin(50 nM), by 5 +/- 1% at 4.8 ms and by 19 +/- 2% at 350 ms, indicating that L-arginine could reduce an apamin-insensitive Ik(Ca)6. Calcium-activated potassium currents were also sensitive to charybdotoxin (10 nM), as this reduced the outward current by 34 +/- 4% at 350 ms when a high calcium-containing pipette was used during a depolarizing command to + 40 mV. L-Arginine still decreased the outward current in the presence of charybdotoxin, by 6 +/- 1% at 4.8 ms and 12 +/- 4% at 350 ms, showing that L-arginine could reduce a charybdotoxin-insensitive Ik(Ca).7. The present results indicate that NO-synthase in ciliary ganglia can modulate Ik(Ca) by a method which is independent of the action of NO on the calcium channels. The Ik(ca) is decreased significantly at 4.8 ms into a depolarizing pulse, at a time that would decrease the rate of repolarization of the action potential. Ik(Ca) is also reduced at longer times (350 ms), indicating an affect on the inactivating process.

Nitric oxide and arachidonic acid modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of high-voltage activated transient and sustained calcium currents in cultured neurones of avian ciliary ganglia by nitric oxide (NO) and arachidonic acid. 2. Sodium nitroprusside (100 microM) reduced the transient calcium current (ICa) on average by 31% and the sustained ICa by 32% during a test depolarization to +20 mV from a holding potential of -100 mV. This reduction was maintained for at least 30 min following a single application of sodium nitroprusside. 3. L-Arginine (270 microM) reduced the transient ICa on average by 28% and the sustained ICa by 22% and these effects were prevented by the presence of the NO-synthase competitive blocker NG-nitro-L-arginine methylester (L-NAME; 100 microM) in the bathing solution. 4. Arachidonic acid (50 microM) reduced the transient ICa on average by 28% and the sustained ICa by 33%. When added together, arachidonic acid (50 microM) and L-arginine (270 microM) produced the same effects as arachidonic acid alone. 5. Blocking the conversion of arachidonic acid to prostaglandins by addition of indomethacin (20 microM) to the bathing solution did not prevent the depression of either the transient or the sustained calcium current during application of arachidonic acid (50 microM). The effects of arachidonic acid were also not occluded by L-NAME (100 microM) when present in the bathing solution. 6. Inhibiting the biosynthesis of leukotrienes by applying L-663,536 (MK-886; 3 microM) to the bathing solution prevented the depression of both components of ICa during application of arachidonic acid (50 microM). 7. These results indicate that endogenous NO and arachidonic acid pathways are present in parasympathetic ciliary neurones, and that both act to depress high-voltage, gated, calcium channel activity.

A sensitive test for studying the effects of opioids on a C-fibre reflex elicited by a wide range of stimulus intensities in the rat.

A C-fibre reflex elicited by electrical stimulation within the receptive field of the sural nerve was recorded from the ipsilateral biceps femoris muscle in anaesthetized rats. Recruitment curves were built by varying the stimulus intensity from 0 to 50 mA and temporal evolution was studied by using a constant level of stimulation. At a constant level of stimulation, intrathecal administration of morphine resulted in a depressive effect on the C-fibre reflex in the 0.18-0.75 microgram range (ED50 = 0.2 microgram). Study of the recruitment curves showed that, in the 0.18-0.375 microgram range, morphine had little effect on the threshold, but induced significant decreases in the slopes. At doses above 0.75 microgram, morphine modified both the threshold and the slope of the recruitment curves. Systemic naloxone totally reversed these effects. It is concluded that intrathecal morphine not only produces a shift in the encoding functions of the spinal cord but also reduces the gain of these functions. It is suggested that this method is reliable for the pharmacological study of the spinal transmission of nociceptive signals.

Tachyphylaxis of the intrinsic cardiac nerves to nicotine: effects on A-V nodal conduction.

1. The possibility that responses of the intrinsic cardiac nerves (ICN) of the dog to nicotinic stimulation are influenced by tachyphylaxis to repeated administration of nicotine (NIC) was evaluated in an anaesthetized preparation. Prolongation of A-V conduction was used as an index of ICN responsiveness. 2. Twenty dogs were placed on cardiopulmonary bypass and an electrode was sutured over the His bundle. Both vagi were sectioned, beta-adrenoceptor blockade instituted, and the hearts were paced. Nicotine (2-100 micrograms) was administered directly into the coronary circulation via an aortic catheter. Tachyphylaxis was estimated from the rate of deterioration of negative dromotropic effect of NIC in response to various protocols of repeated doses. 3. Tachyphylaxis was not observed in response to repeated doses of acetylcholine. 4. Tachyphylaxis to nicotine was found to be both time- and dose-dependent; i.e. increasing the dose or decreasing the time between doses to less than 3 min augmented its development. 5. Tachyphylaxis was pronounced after 5 x 100 micrograms doses of NIC, even if the test doses were greater than 3 min apart and 20 min was allowed to elapse between the two series of test doses. 6. These data are descriptive in nature and no mechanism for the tachyphylaxis could be deduced. Pharmacokinetic data of this nature have not been previously reported for nicotinic stimulation of the intrinsic parasympathetic ganglia of the canine heart. Tachyphylaxis can be avoided when testing the intrinsic innervation of the canine heart if the doses used are less than 100 micrograms and are administered at least 3 min apart.

Prejunctional opioid mu-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors.

Experiments were designed to study the interaction between prejunctional alpha 2-adrenoceptors and both adenosine and opioid receptors at the postganglionic sympathetic nerve endings innervating the tail artery of the rat. Segments of this vessel were preincubated with [3H]-noradrenaline and then perfused/superfused with [3H]-noradrenaline-free medium. Their perivascular nerves were field stimulated with standard stimulation parameters: 24 pulses at 0.4 Hz, 0.3 ms, 200 mA. In some experiments, the stimulation parameters were adjusted in order to obtain similar reference release values despite the presence of a first release-modulating drug. The adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.3-10 mumol/l) and [D-Ala2,MePhe4,Glyol5]enkephalin (DAGO; 0.3-10 mumol/l) depressed the stimulation-evoked overflow of tritium in a concentration dependent manner. The release-inhibiting effect of both NECA and DAGO was enhanced in the presence of the alpha 2-adrenoceptor antagonist rauwolscine (3 mumol/1) while it was attenuated in the presence of the alpha 2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK-14,304; 0.1 mumol/l). These changes occurred both at standard and adjusted stimulation parameters. These results demonstrate that the prejunctional adenosine A1- and opioid mu-receptors interact with the prejunctional alpha 2-adrenoceptors. The level at which these interactions take place (receptors themselves or transduction mechanisms) as well as the physiological significance of the phenomenon remain to be determined.

Albumin inhibits effects of Cu2+ on autonomic postganglionic transmission of guinea-pig ileum and vas deferens.

The influence of albumin, 4.5 and 45 g/l, on the effects of Cu2+, 10(-9)-10(-3) M, on the neuromuscular transmission of the isolated guinea-pig ileum and vas deferens was investigated. Low concentrations of Cu2+, 10(-9)-10(-6) M, caused a slight and transient increase of the contractile response to direct muscle stimulation of the vas deferens. Albumin at 4.5 g/l inhibited this effect. The basal tone of the ileum increased transiently when Cu2+ was added in the presence of albumin, 4.5 g/l. A stimulant action of Cu2+, 10(-9)-10(-6) M, was seen on both organs, when contractions were induced by nerve stimulation. All these effects were inhibited by albumin, 45 g/l. Higher concentrations of Cu2+, 10(-5)-10(-4) M, increased the basal tone of the ileum and the vas deferens. In the presence of albumin, 45 g/l, this stimulating effect of Cu2+ appeared in higher concentrations. The results suggest that Cu2+ is more likely to influence the function of neuronal tissues when the concentration of protein is low.

Neuropeptide-Y innervation of the rat spleen: another potential immunomodulatory neuropeptide.

Neuropeptide-Y (NPY) is a 36 amino acid peptide that acts as a chemical messenger in the central and peripheral nervous systems. NPY often is found colocalized with the classical neurotransmitter norepinephrine (NE) and can potentiate the effects of this neurotransmitter postsynaptically in many systems. Using immunocytochemistry for NPY and specific lymphoid cell markers, we mapped the distribution of NPY-positive nerve fibers in the rat spleen. NPY-positive nerve fibers were present along the vasculature, trabeculae, and capsule, and also were found associated with specific lymphoid parenchymal compartments of the spleen, in close contact with lymphocytes and macrophages. These contacts were investigated further at the electron microscopic level. NPY-positive nerve terminals were found in close apposition with lymphocytes in the periarteriolar lymphatic sheath, and with lymphocytes and macrophages in the marginal zone. Previous studies have reported that postganglionic noradrenergic nerve fibers innervate specific lymphoid compartments of the rat spleen, with nerve terminals forming direct appositions with cells of the immune system. The possible colocalization of NPY and NE in these nerve fibers was investigated by chemical sympathectomy with 6-hydroxydopamine, followed by immunocytochemical labeling of NPY and tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine synthesis. Colocalization also was investigated by labeling for NPY with a fluorescent label, eluting the NPY, and staining for TH with diaminobenzidine as the label. These studies demonstrate that norepinephrine and NPY are colocalized in the postganglionic sympathetic nerve fibers of the rat spleen.(ABSTRACT TRUNCATED AT 250 WORDS)