Vascular-dependent effects of elevated glucose on postganglionic sympathetic neurons

Perivascular sympathetic nerves are important determinants of vascular function that are likely to contribute to vascular complications associated with hyperglycemia and diabetes. The present study tested the hypothesis that glucose modulates perivascular sympathetic nerves by studying the effects of 7 days of hyperglycemia on norepinephrine (NE) synthesis [tyrosine hydroxylase (TH)], release, and uptake. Direct and vascular-dependent effects were studied in vitro in neuronal and neurovascular cultures. Effects were also studied in vivo in rats made hyperglycemic (blood glucose >296 mg/dl) with streptozotocin (50 mg/kg). In neuronal cultures, TH and NE uptake measured in neurons grown in high glucose (HG; 25 mM) were less than that in neurons grown in low glucose (LG; 5 mM) (P < 0.05; n = 4 and 6, respectively). In neurovascular cultures, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release from neurovascular cultures grown in HG (1.8 ± 0.2%; n = 5) was greater than that from cultures grown in LG (0.37 ± 0.28%; n = 5; P < 0.05; unpaired t-test). In vivo, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release in hyperglycemic animals (9.4 + 1.1%; n = 6) was greater than that in control animals (5.39 + 1.1%; n = 6; P < 0.05; unpaired t-test). These data identify a novel vascular-dependent effect of elevated glucose on postganglionic sympathetic neurons that is likely to affect the function of perivascular sympathetic nerves and thereby affect vascular function.     

Preganglionic discharge induced by acetylcholine in the superior cervical ganglia of the rat

ACh (5.10(-4) M), when applied to isolated ganglion preparations elicited an apparently antidromic discharge in the cervical sympathetic trunk. The intensity of this back-firing was found to be about 10 times lower than that of the postganglionic discharge evoked by ACh in the internal carotid nerve. Both responses however displayed a similar time course consisting mainly of an early and a late component. In the back-firing the early component died out in few seconds, while the late one lasted 20-30 seconds. The two components were cancelled by d-tubocurarine (5.10(-6) M) and atropine (10(-6) M) respectively, suggesting that both nicotinic and muscarinic cholinoceptive sites are involved. In chronically decentralized preparations ACh evoked a clear back-firing response not substantially different from that elicited in normal ganglia. Therefore it is likely that the back-firing phenomenon is not due to antidromic activation of preganglionic fibers. The back-firing observed in the rat superior cervical ganglion was interpreted as being due to activation of sympathetic neurons, known to give rise to recurrent axons in the cervical sympathetic cord.     

Musings on the wanderer: what's new in our understanding of vago-vagal reflex? IV. Current concepts of vagal efferent projections to the gut

Vagal efferents, consisting of distinct lower motor and preganglionic parasympathetic fibers, constitute the motor limb of vagally mediated reflexes. Arising from the nucleus ambiguus, vagal lower motor neurons (LMN) mediate reflexes involving striated muscles of the orad gut. LMNs provide cholinergic innervation to motor end plates that are inhibited by myenteric nitrergic neurons. Preganglionic neurons from the dorsal motor nucleus implement parasympathetic motor and secretory functions. Cholinergic preganglionic neurons form parallel inhibitory and excitatory vagal pathways to smooth muscle viscera and stimulate postganglionic neurons via nicotinic and muscarinic receptors. In turn, the postganglionic inhibitory neurons release ATP, VIP, and NO, whereas the excitatory neurons release ACh and substance P. Vagal motor effects are dependent on the viscera's intrinsic motor activity and the interaction between the inhibitory and excitatory vagal influences. These interactions help to explain the physiology of esophageal peristalsis, gastric motility, lower esophageal sphincter, and pyloric sphincter. Vagal secretory pathways are predominantly excitatory and involve ACh and VIP as the postganglionic excitatory neurotransmitters. Vagal effects on secretory functions are exerted either directly or via release of local mediators or circulating hormones.     

The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons

1. The ramped voltage clamp technique was developed as a rapid means of studying the effects of certain nicotinic and muscarinic agents on ionic involvement and conductance changes during acetylcholine (ACh) responses of Helix pomatia neurons. 2. Atropine was found to be a potent cholinolytic on A-type neurons, ACh responses of which are blocked by ouabain and mediated by Na+ and Cl- permeabilities, while d-tubocurarine blocked B-type ACh responses which are insensitive to ouabain and mediated by Na+ and K+ permeabilities. 3. Nicotinic agent butyrylcholine was found to be a potent cholinomimetric on B-type cells. 4. The results suggest that ACh receptors on A-type cells are more "muscarinic" while those on B-type cells are more "nicotinic". 5. It was also suggested that both muscarinic and nicotinic ACh receptors may coexist in the Helix neuronal membrane and the possibility of ACh interacting with one of them is determined by the level of phosphorylation of the membrane proteins.     

Hydrolysis, synthesis, and release of acetylcholine in the isolated heart

The occurrence of unhydrolyzed acetylcholine (ACh) in the cardiac perfusate during vagal stimulation in the absence of cholinesterase inhibition has been demonstrated by several methods. Because some ACh was found unhydrolyzed in the extracellular space for several seconds after vagal stimulation (half-time of decay 2.5 s), it appears that the prolonged time course of the cardiac responses to bursts of vagal activity is determined by a slow rate of transmitter inactivation (diffusion plus hydrolysis) in addition to slowly operating postsynaptic mechanisms mediated by activation of the muscarinic receptor. The neuronal uptake of choline in isolated heart preparations was found to be Na+ dependent, sensitive to hemicholinium 3, and activated by vagal stimulation. Activation occurred after a delay of 1 or 2 min and slowly faded within 5 min after stimulation. Resting release of ACh was insensitive to extracellular Ca2+ and to muscarinic feedback inhibition, in contrast to the evoked transmitter release. Inasmuch as atropine increased ACh release by vagal and field stimulation to the same extent, muscarinic feedback inhibition is likely to occur at postganglionic parasympathetic neurons. Adrenergic agonists and propranolol did not significantly change the release of ACh.     

Immunosuppressive effects of a Kv1.3 inhibitor

The voltage gated potassium channel (Kv1.3) has been shown to play a role in immune responsiveness. Blockade of the channel led to diminution of T cell activation and delayed type hypersensitivity. Previous in vitro studies of the blockade were focused on T cell activation and proliferation. In this study we examined other T and monocytic cell mediated events to glean the extent of the immunosuppressive effects of a Kv1.3 specific inhibitor, Margatoxin (MgTX). We found that MgTX inhibited the intracellular production of Th-1 as well as Th-2 cytokines. MgTX can also inhibit IL-2 production and proliferation of T cells upon stimulation with anti-CD3 and VCAM-1. Furthermore, a redirected cytolytic activity was also inhibited by MgTX. However, MgTX did not inhibit generation of CTL to EBV transformed lymphoma cells or antibody-dependent cellular cytolysis mediated by monocytes. It appears that a Kv1.3 blockade does not affect all immune responses, particularly those of innate immunity.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.     

Colonic soluble mediators from the maternal separation model of irritable bowel syndrome activate submucosal neurons via an interleukin-6-dependent mechanism

Irritable bowel syndrome (IBS) is characterized by episodic bouts of abdominal pain, bloating, and altered bowel habit. Accumulating evidence has linked immune activation with IBS, including reports of increases in circulating levels of the proinflammatory cytokine interleukin (IL)-6. However, it is unknown whether IL-6 contributes directly to disease manifestation. As enteric nervous activity mediates motility and secretory function, the aims of this study were to determine the effects of IL-6 on submucosal neurons and related gastrointestinal (GI) function. In these studies, we examined the colons of maternally separated (MS) rats, which exhibit elevated circulating levels of IL-6 in addition to GI dysfunction. To our knowledge, these studies are the first to provide evidence of the sensitivity of submucosal neurons to colonic secretions from MS rats (n = 50, P < 0.05), thus recapitulating clinical biopsy data. Moreover, we demonstrated that the excitatory action is IL-6 dependent. Thereafter, the impact of IL-6 on neuronal and glial activation and absorpto/secretory function was pharmacologically characterized. Other proinflammatory cytokines including IL-8 (n = 30, P > 0.05), IL-1β (n = 56, P > 0.05), and TNF-α (n = 56, P > 0.05) excited fewer neurons. Both muscarinic and nicotinic cholinergic receptors participate in the effect and cause downstream activation of ERK, JAK-STAT, and NF-κB signaling cascades. Functionally, IL-6 increases transepithelial resistance and enhances neurally and cholinergically mediated ion transport. These data provide a role for IL-6 in colonic secretory functions and relate these effects to GI dysfunction in an animal model of IBS, thereby elucidating a potential relationship between circulating levels of IL-6 and aberrant GI function.     

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Effects of various kinds of stimulation on ornithine decarboxylase activity in superior cervical sympathetic and nodose ganglia of rats

Levels of cyclic nucleotides and ornithine decarboxylase (ODC) activity were examined following the application of various kinds of stimuli to superior cervical sympathetic ganglia (SCG), nodose ganglia, and vagus nerve fibers excised from the rat. The level of cyclic GMP in the SCG rose rapidly to about 4.5- to 7.5-fold the unstimulated control with 10 min of incubation after applications of preganglionic electrical stimulation (10 Hz), acetylcholine (ACh; 1 mM), or high extracellular K+ ( [K+]0, 70 mM). The cyclic GMP level in nodose ganglia was increased less than in the SCG by either ACh or high [K+]0 but was not affected by ACh in vagus fibers. Cyclic AMP in the SCG was also increased about 4- to 5.5-fold over the control within 10 min with the addition of ACh, norepinephrine (NE; 0.05 mM), or high [K+]0. Although NE caused a small increase in cyclic AMP, neither ACh nor high [K+]0 produced any appreciable change in nodose ganglia or vagus fibers. The ODC activity in the SCG was increased by preganglionic stimulation of 3- to 4-hr duration but not by a shorter period. A similar change in ODC activity was caused by the addition of oxotremorine (1 mM), isoproterenol (0.1 mM), NE, cyclic AMP (1 mM), or dibutyryl cyclic GMP (1 mM). The effect was exaggerated by the further addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. The increase in ODC activity caused by ACh was abolished by a muscarinic cholinergic antagonist, atropine (0.01 mM), and following axotomy for a week, but not by a nicotinic antagonist or by denervation in the SCG. A similar increase in ganglionic ODC activity by NE was inhibited by an adrenergic blocker, propranolol (0.01 mM), and following axtotomy for a week, but not by denervation. Cholinergic or adrenergic stimulation did not cause an increase in ODC activity in nodose ganglia or vagus fibers. These results suggest that the stimulation-induced increase in ODC activity occurs in postganglionic neurons rather than in satellite glial cells and is mediated by muscarinic cholinergic or adrenergic receptors. The process appears to involve cyclic nucleotide-mediated protein biosynthesis in the SCG.     

Hypothermia reduces ischemia- and stimulation-induced myocardial interstitial norepinephrine and acetylcholine releases.

Although hypothermia is one of the most powerful modulators that can reduce ischemic injury, the effects of hypothermia on the function of the cardiac autonomic nerves in vivo are not well understood. We examined the effects of hypothermia on the myocardial interstitial norepinephrine (NE) and ACh releases in response to acute myocardial ischemia and to efferent sympathetic or vagal nerve stimulation in anesthetized cats. We induced acute myocardial ischemia by coronary artery occlusion. Compared with normothermia (n = 8), hypothermia at 33 degrees C (n = 6) suppressed the ischemia-induced NE release [63 nM (SD 39) vs. 18 nM (SD 25), P < 0.01] and ACh release [11.6 nM (SD 7.6) vs. 2.4 nM (SD 1.3), P < 0.01] in the ischemic region. Under hypothermia, the coronary occlusion increased the ACh level from 0.67 nM (SD 0.44) to 6.0 nM (SD 6.0) (P < 0.05) and decreased the NE level from 0.63 nM (SD 0.19) to 0.40 nM (SD 0.25) (P < 0.05) in the nonischemic region. Hypothermia attenuated the nerve stimulation-induced NE release from 1.05 nM (SD 0.85) to 0.73 nM (SD 0.73) (P < 0.05, n = 6) and ACh release from 10.2 nM (SD 5.1) to 7.1 nM (SD 3.4) (P < 0.05, n = 5). In conclusion, hypothermia attenuated the ischemia-induced NE and ACh releases in the ischemic region. Moreover, hypothermia also attenuated the nerve stimulation-induced NE and ACh releases. The Bezold-Jarisch reflex evoked by the left anterior descending coronary artery occlusion, however, did not appear to be affected under hypothermia.     

An AF-DX 116 sensitive inhibitory mechanism modulates nicotinic and muscarinic transmission in cat superior cervical ganglion in the presence of anticholinesterase.

The effect of the muscarinic receptor antagonist AF-DX 116 on the inhibitory action of muscarinic agonists and on responses mediated by nicotinic or muscarinic ganglionic transmission was studied in the superior cervical ganglion of the anesthetized cat. The postganglionic compound action potential evoked by cervical sympathetic trunk stimulation was depressed by methacholine or acetylcholine (ACh) injected into the ganglionic arterial supply. The depression was blocked by AF-DX 116. The compound action potentials evoked by preganglionic stimulus trains were also depressed when the intratrain frequency was 2 Hz or greater. This intratrain depression was, however, insensitive to AF-DX 116. The anticholinesterase drug physostigmine markedly enhanced the intratrain depression of the compound action potential. This effect was reversed by AF-DX 116. During nicotinic receptor block with hexamethonium, preganglionic stimulus trains with intratrain frequencies of 5 Hz or greater produced nicitating membrane contractions that could be blocked by the M1 muscarinic receptor antagonist pirenzepine. The amplitude of the contractions increased with frequency and reached a maximum at 20-40 Hz. AF-DX 116 had no effect on these responses. After administration of physostigmine, the amplitude of the nictitating membrane responses decreased with increasing intratrain frequency. AF-DX 116 reversed this effect. The data suggest that, in the superior cervical ganglion, AF-DX 116 sensitive muscarinic receptors which depress synaptic transmission are activated by exogenous agonists but not by the ACh released by the preganglionic axon terminals unless cholinesterase activity is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

CSF acidosis augments ventilation through cholinergic mechanisms

Ventilation is influenced by the acid-base status of the brain extracellular fluids (ECF). CO2 may affect ventilation independent of changes in H+. Whether the acidic condition directly alters neuronal firing or indirectly alters neuronal firing through changes in endogenous neurotransmitters remains unclear. In this work, ventriculocisternal perfusion (VCP) was used in anesthetized (pentobarbital sodium, 30 mg/kg) spontaneously breathing dogs to study the ventilatory effects of acetylcholine (ACh), eucapnic acidic (pH approximately 7.0) cerebrospinal fluid (CSF), and hypercapnic acidic (pH approximately 7.1) CSF in the absence and presence of atropine (ATR). Each animal served as its own control. Base line was defined during VCP with control mock CSF (pH approximately 7.4). With ATR (4.8 mM) there was an insignificant downward trend in minute ventilation (VE). ACh (6.6 mM) increased VE 53% (n = 12, P less than 0.01), eucapnic acidic CSF increased VE 41% (n = 12, P less than 0.01), and hypercapnic acidic CSF increased VE 47% (n = 6, P less than 0.01). These positive effects on ventilation were not seen in the presence of ATR. This suggests that acidic brain ECF activates ventilatory neurons through muscarinic cholinergic mechanisms. Higher concentrations of ACh increased ventilation in a concentration-dependent manner. Higher concentrations of ATR decreased ventilation progressively, resulting in apnea. The results suggest that ACh plays a significant role in the central augmentation of ventilation when the brain ECF is made acidic by either increasing CSF PCO2 or decreasing CSF bicarbonate.     

Cholinergic neurotransmission from mechanosensory afferents to giant interneurons in the terminal abdominal ganglion of the cricket Gryllus bimaculatus

Crickets respond to air currents with quick avoidance behavior. The terminal abdominal ganglion (TAG) has a neuronal circuit for a wind-detection system to elicit this behavior. We investigated neuronal transmission from cercal sensory afferent neurons to ascending giant interneurons (GIs). Pharmacological treatment with 500 muM acetylcholine (ACh) increased neuronal activities of ascending interneurons with cell bodies located in the TAG. The effects of ACh antagonists on the activities of identified GIs were examined. The muscarinic ACh antagonist atropine at 3-mM concentration had no obvious effect on the activities of GIs 10-3, 10-2, or 9-3. On the other hand, a 3-mM concentration of the nicotinic ACh antagonist mecamylamine decreased spike firing of these interneurons. Immunohistochemistry using a polyclonal anti-conjugated acetylcholine antibody revealed the distribution of cholinergic neurons in the TAG. The cercal sensory afferent neurons running through the cercal nerve root showed cholinergic immunoreactivity, and the cholinergic immunoreactive region in the neuropil overlapped with the terminal arborizations of the cercal sensory afferent neurons. Cell bodies in the median region of the TAG also showed cholinergic immunoreactivity. This indicates that not only sensory afferent neurons but also other neurons that have cell bodies in the TAG could use ACh as a neurotransmitter.     

Neuronal control of heart rate in isolated mouse atria

A novel mouse isolated atrial preparation with intact postganglionic autonomic innervation was used to investigate the neuronal control of heart rate. To establish whether autonomic activation was likely to alter heart rate by modulating the hyperpolarization-activated current (If), the L-type Ca2+ current (ICa,L), or the ACh-activated K+ current (IK,ACh), the effects of nerve stimulation (right stellate ganglion or right vagus, 1-30 Hz) and autonomic agonists (0.1 microM norepinephrine or 0.3 microM carbachol) on heart rate were investigated in the presence of inhibitors of these currents, cesium chloride (Cs+, 1 mM), nifedipine (200 nM), and barium chloride (Ba2+, 0.1 mM), respectively. The positive chronotropic response to stellate ganglion stimulation was reduced by approximately 20% with Cs+ and nifedipine (P < 0.05), whereas the heart rate response to norepinephrine was only reduced with Cs+ (P < 0.05). Ba2+ attenuated the decrease in heart rate with vagal stimulation and carbachol by approximately 60% (P < 0.05). These results are consistent with the idea that sympathetic nerve stimulation modulates If to increase heart rate in the mouse. Activation of ICa,L also appears to contribute to the sympathetic heart rate response. However, the decrease in heart rate with vagal stimulation or carbachol is likely to result primarily from the activation of IK,ACh.     

Cholinergic Pairing with Visual Activation Results in Long-Term Enhancement of Visual Evoked Potentials

Acetylcholine (ACh) contributes to learning processes by modulating cortical plasticity in terms of intensity of neuronal activity and selectivity properties of cortical neurons. However, it is not known if ACh induces long term effects within the primary visual cortex (V1) that could sustain visual learning mechanisms. In the present study we analyzed visual evoked potentials (VEPs) in V1 of rats during a 4–8 h period after coupling visual stimulation to an intracortical injection of ACh analog carbachol or stimulation of basal forebrain. To clarify the action of ACh on VEP activity in V1, we individually pre-injected muscarinic (scopolamine), nicotinic (mecamylamine), α7 (methyllycaconitine), and NMDA (CPP) receptor antagonists before carbachol infusion. Stimulation of the cholinergic system paired with visual stimulation significantly increased VEP amplitude (56%) during a 6 h period. Pre-treatment with scopolamine, mecamylamine and CPP completely abolished this long-term enhancement, while α7 inhibition induced an instant increase of VEP amplitude. This suggests a role of ACh in facilitating visual stimuli responsiveness through mechanisms comparable to LTP which involve nicotinic and muscarinic receptors with an interaction of NMDA transmission in the visual cortex.     

ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用

利用全细胞膜片钳技术研究乙酰胆碱(acetylcholine,ACh)对大鼠皮层体感区神经元延迟整流钾电流(IK)的调制作用。结果表明:(1)ACh(0．1、1、10、100 μmol/L)对大鼠皮层体感区神经元IK有抑制作用,并具有剂量依赖性关系(P<0．01)。 (2)ACh可使IK激活曲线的斜率变大,并使激活曲线向超极化方向移动。IK激活曲线的半数激活电压(V1/12)和斜率因子(k)分别由给药前的(-41．8±9．7)mV和(30．7±7．2)mV变为给药后的(-122．4±38．6)mV和(42．4±7．0)mV。(3)100 μmol/L的N受体拮抗剂筒箭毒碱(tubocurarine)可减弱ACh对IK的抑制作用,在指令电压+60 mV时tubocurarine+ACh组的IK幅度下降了(16．9± 13．8)％(n=8),与10 μmol/L ACh组引起的(36．5±7．8)％的IK下降幅度相比,有极显著差异(P<0．01)。10 μmol/L的M1受体拮抗剂哌仑西平(pirenzepin)拮抗ACh对IK的抑制作用不明显(n=7,P>0．05);而10 μmol/L的M3受体拮抗剂4-DAMP可部分拮抗ACh对IK的抑制作用,并且4-DAMP+ACh组使IK的电流值下降了(26．8±4．7)％(n=6),与ACh组引起的IK电流下降相比,有显著差异(P<0．05)。(4)蛋白激酶C(protein kinase C,PKC)阻断剂chelerythrine拮抗ACh对IK的抑制作用,PKC激动剂PDBu可增强ACh对IK的抑制作用(P<0．05)。综上所述,ACh对人鼠皮层体感区神经元IK的抑制作用主要是通过烟碱受体(nAChRs)和M3受体介导,并经过PKC信号途径。     

Vascular-derived artemin: a determinant of vascular sympathetic innervation?

Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor alpha3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 +/- 11.9 mm, and that in the absence of FA was 26.3 +/- 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.