Craig B. Neylon

Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (KCa) and connexins: possible relationship to vasodilator function?

Activation of endothelial cell small‐ (S) and intermediate‐ (I) conductance calcium‐activated potassium channels (KCa) and current or molecular transfer via myoendothelial gap junctions underlies endothelium‐derived hyperpolarization leading to vasodilation. The mechanism underlying the KCa component of vasodilator activity and the characteristics of gap junctions are targets for the selective control of vascular function. In the rat mesenteric artery, where myoendothelial gap junctions and connexin (Cx) 40 are critical for the transmission of the endothelial cell hyperpolarization to the smooth muscle, SKCa and IKCa provide different facets of the endothelium‐derived hyperpolarization response, being critical for the hyperpolarization and repolarization phases, respectively. The present study addressed the question of whether this functional separation of responses may be related to the spatial localization of the associated channels? The distribution of endothelial SKCa and IKCa and Cx subtype(s) were examined in the rat mesenteric artery using conventional confocal and high‐resolution ultrastructural immunohistochemistry. At the internal elastic lamina–smooth muscle cell interface at internal elastic lamina holes (as potential myoendothelial gap junction sites), strong punctate IKCa, Cx37 and Cx40 expression was present. SKCa, Cx37, Cx40 and Cx43 were localized to adjacent endothelial cell gap junctions. High‐resolution immunohistochemistry demonstrated IKCa and Cx37‐conjugated gold to myoendothelial gap junction‐associated endothelial cell projections. Clear co‐localization of KCa and Cxs suggests a causal relationship between their activity and the previously described differential functional activation of SKCa and IKCa. Such precise localizations may represent a selective target for control of vasodilator function and vascular tone.

Evidence for Involvement of Both IKCa and SKCa Channels in Hyperpolarizing Responses of the Rat Middle Cerebral Artery

Background and Purpose— Endothelium-derived hyperpolarizing factor responses in the rat middle cerebral artery are blocked by inhibiting IKCa channels alone, contrasting with peripheral vessels where block of both IKCa and SKCa is required. As the contribution of IKCa and SKCa to endothelium-dependent hyperpolarization differs in peripheral arteries, depending on the level of arterial constriction, we investigated the possibility that SKCa might contribute to equivalent hyperpolarization in cerebral arteries under certain conditions. Methods— Rat middle cerebral arteries (≈175 &mgr;m) were mounted in a wire myograph. The effect of KCa channel blockers on endothelium-dependent responses to the protease-activated receptor 2 agonist, SLIGRL (20 &mgr;mol/L), were then assessed as simultaneous changes in tension and membrane potential. These data were correlated with the distribution of arterial KCa channels revealed with immunohistochemistry. Results— SLIGRL hyperpolarized and relaxed cerebral arteries undergoing variable levels of stretch-induced tone. The relaxation was unaffected by specific inhibitors of IKCa (TRAM-34, 1 &mgr;mol/L) or SKCa (apamin, 50 nmol/L) alone or in combination. In contrast, the associated smooth-muscle hyperpolarization was inhibited, but only with these blockers in combination. Blocking nitric oxide synthase (NOS) or guanylyl cyclase evoked smooth-muscle depolarization and constriction, with both hyperpolarization and relaxation to SLIGRL being abolished by TRAM-34 alone, whereas apamin had no effect. Immunolabeling showed SKCa and IKCa within the endothelium. Conclusions— In the absence of NO, IKCa underpins endothelium-dependent hyperpolarization and relaxation in cerebral arteries. However, when NOS is active SKCa contributes to hyperpolarization, whatever the extent of background contraction. These changes may have relevance in vascular disease states where NO release is compromised and when the levels of SKCa expression may be altered.

Different electrical responses to vasoactive agonists in morphologically distinct smooth muscle cell types.

Vascular smooth muscle cells (SMCs) in the blood vessel wall are frequently heterogeneous in nature, differing in their gross morphology, size, and shape, subcellular organelles, cytoskeleton, and contractile protein composition. In adult rat arterial vessels, two populations of SMCs have been shown to predominate: elongated bipolar cells, representing the majority of cells, and epithelial-like SMCs. We examined the ionic responses of these two types of SMCs, isolated by multiple subculture, to vasoactive stimuli. Elevations in intracellular Na+ and Ca2+ were measured with SBFI and fura 2, respectively, and changes in membrane potential were measured using the potential-sensitive fluorescent probe bis-oxonol. The resting membrane potential of the elongated bipolar cells was less negative than that of the epithelial-like SMCs. Exposure of the elongated SMCs to endothelin 1, alpha-thrombin, or arginine vasopressin induced elevations in [Ca2+]i and [Na+]i and membrane depolarization. Depolarization occurred because of entry of both Na+ and Ca2+, and pharmacological blockade of Cl- or K+ channels did not attenuate the depolarization. In contrast, when [Ca2+]i was elevated by the same agonists in the epithelial-like SMCs there was a pronounced hyperpolarization that appeared to be the consequence of enhanced activity of charybdotoxin-sensitive Ca(2+)-activated K+ channels because it was abolished by charybdotoxin (20 nmol/L), partially attenuated by tetraethylammonium chloride (10 mmol/L), and unaffected by apamin (1 mumol/L), glibenclamide (1 mumol/L), or 4-aminopyridine (5 mmol/L). Chelation of [Ca2+]i also abolished the hyperpolarization; instead, a small depolarization was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]-rauwolscine binding to alpha 2-adrenoceptors in the mammalian kidney: apparent receptor heterogeneity between species.

1 Binding of the α2‐adrenoceptor antagonist [3H]‐rauwolscine was characterized in membrane preparations from the kidneys of mouse, rat, rabbit, dog, and man. 2 In all species, binding reached equilibrium within 45 min and dissociated at a single exponential rate after addition of phentolamine 10 μM. 3 Saturation studies showed that the affinity of [3H]‐rauwolscine was similar in all species (2.33‐3.03 nM) except man where it was significantly higher (0.98 nM). Marked differences were seen in the density of binding sites, increasing in the order: man < dog < rabbit < rat < mouse. In all cases, Hill coefficients were not significantly different from unity. 4 [3H]‐rauwolscine binds with low affinity (KD > 15 nM) to membranes prepared from guinea‐pig kidney. The low affinity binding is not due to the absence of particular ions in the incubation medium or to receptor occupation by endogenous agonist. 5 The binding in all species was found to be stereoselective with respect to the isomers of noradrenaline. However, differences were seen in the characteristics of agonist interactions with the binding site both between isomers and between species. 6 Marked differences in affinity of particular α‐adrenoceptor antagonists were observed for α2‐adrenoceptors labelled by [3H]‐rauwolscine. These differences were most evident with the α1‐adrenoceptor selective antagonist prazosin which displayed inhibition constants (Ki values) of 33.2, 39.5, 261, 570 and 595 nM in rat, mouse, dog, man and rabbit, respectively. 7 Differences are apparent in the characteristics of α2‐adrenoceptors labelled by [3H]‐rauwolscine between species and it is suggested that the differences observed for α1‐selective antagonists such as prazosin may be related to binding to additional sites in the vicinity of the α2‐adrenoceptor.

Stimulation of α1‐adrenoceptors in rat kidney mediates increased inositol phospholipid hydrolysis

1 The molecular events which follow activation of α1‐adrenoceptors in rat kidney were investigated by measuring inositol phospholipid hydrolysis. Slices were labelled with [3H]‐inositol (0.25 μm) and the accumulation of [3H]‐inositol phosphates ([3H]‐IPs) was measured after stimulation with α‐adrenoceptor agonists. 2 Phospholipid labelling was both time‐and Ca2+‐dependent. In kidney, Ca2+ (1 mm) increased the incorporation of [3H]‐inositol by 49% and in cerebral cortex reduced it by 46%. 3 Following addition of noradrenaline (NA, 1 mm), accumulation of [3H]‐IPs increased linearly for at least 60 min. In Ca2+‐free buffers a 2.1 fold increase in [3H]‐IP accumulation was observed and further increases in stimulated and control levels were produced in the presence of Ca2+ (2.5 mm). These responses were attenuated by the inclusion of indomethacin (10 μm) and abolished in the presence of EGTA (0.5 mm). Responses to (−)‐NA were more than 4 fold higher in the renal cortex than in the medulla. 4 Separation of the IPs which accumulate after α‐adrenoceptor agonists showed that after 60 min stimulation the major products were glycerophosphoinositol and inositol‐phosphate with smaller amounts of inositol‐bisphosphate and inositol‐trisphosphate. 5 The most effective agonists tested for stimulation of accumulation of [3H]‐IPs were (–)‐ NA > phenylephrine > methoxamine, (+)‐NA. Clonidine and (–)‐isoprenaline were ineffective at concentrations up to 100 μm. The order of effectiveness of α‐adrenoceptor antagonists was prazosin > BE2254 > phentolamine > idazoxan > rauwolscine. 6 The results indicate that α1‐adrenoceptors in rat kidney are linked to phosphoinositide hydrolysis and that this response is localized mainly to the renal cortex.

Expression of intermediate conductance potassium channel immunoreactivity in neurons and epithelial cells of the rat gastrointestinal tract

Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.

VASCULAR BIOLOGY OF ENDOTHELIN SIGNAL TRANSDUCTION

1. Endothelins regulate cell function by interacting with two classes of cell surface receptors, ETA and ETB receptors. Both receptor types are members of the heptahelical transmembrane‐spanning receptor superfamily and couple via G‐proteins to multiple intracellular effectors.

Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

Potassium channels and vascular proliferation

Potassium channels are currently the focus of much attention because of their recently discovered role in the regulation of vascular smooth muscle growth. Dramatic alterations in the expression and activity of K+ channels causing marked changes in the cells electrical properties accompany enhanced growth of smooth muscle cells (SMCs). These findings indicate that alterations in K+ channel function are important for SMC proliferation. However, the mechanisms by which changes in K+ channel activity influence cellular growth pathways are poorly understood. The emergent electrical properties caused by modulation of K+ channels are associated with marked differences in the spatial and temporal organization of Ca2+ signaling. Thus, changes in K+ channel function may represent a universal mechanism by which Ca2+ signals are targeted towards activation of gene expression and cell growth. As enhanced growth of smooth muscle underlies many cardiovascular diseases and clinical pathologies, the identification of an important role for K+ channels in SMC proliferation indicates a new source of therapeutic targets to regulate proliferative vascular disorders.

Intermediate-conductance calcium-activated potassium channels in enteric neurones of the mouse: pharmacological, molecular and immunochemical evidence for their role in mediating the slow afterhyperpolarization

Calcium‐activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate‐conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non‐excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT–PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.