Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition.

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

Escherichia coli-mediated impairment of ureteric contractility is uropathogenic E. coli specific.

BACKGROUND: Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. METHODS: Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. RESULTS: Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. CONCLUSIONS: We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved.

Modulation of ureteric Ca signaling and contractility in humans and rats by uropathogenic E. coli.

Ascending urinary tract infections, a significant cause of kidney damage, are predominantly caused by uropathogenic Escherichia coli (UPEC). However, the role and mechanism of changes in ureteric function during infection are poorly understood. We therefore investigated the effects of UPEC on Ca signaling and contractions in rat (n = 17) and human (n = 6) ureters. Ca transients and force were measured and effects of UPEC on the urothelium were monitored in live tissues. In both species, luminal exposure of ureters to UPEC strains J96 and 536 caused significant time-dependent decreases in phasic and high K depolarization-induced contractility, associated with decreases in the amplitude and duration of the Ca transients. These changes were significant after 3-5 h and irreversible over the next 5 h. The infection causes increased activity of K channels, causing inhibition of voltage-gated Ca entry, and K channel blockers could reverse the effects of UPEC on ureteric function. A smaller direct effect on Ca entry also occurs. Nonpathogenic E. coli (TG2) or abluminal application of UPEC did not produce changes in Ca signaling or contractility. UPEC exposure also caused significant impairment of urothelial barrier function; luminal application of the Ca channel blocker nifedipine caused a reduction in contractions as it entered the tissue, an effect not observed in untreated ureters. Thus, UPEC impairs ureteric contractility in a Ca-dependent manner, largely caused by stimulation of potassium channels and this mechanism is dependent on host-urothelium interaction.

Morphology, calcium signaling and mechanical activity in human ureter.

PURPOSE: We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. MATERIALS AND METHODS: Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. RESULTS: Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. CONCLUSIONS: To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.

Airway smooth muscle dysfunction precedes teratogenic congenital diaphragmatic hernia and may contribute to hypoplastic lung morphogenesis.

Fetal intervention aims to improve lung growth and survival in congenital diaphragmatic hernia (CDH). Airway smooth muscle (ASM) is important in lung development: ASM progenitors produce a key growth factor for lung morphogenesis (fibroblast growth factor 10); ASM contractility is also coupled to growth. ASM hyperreactivity occurs in postnatal CDH and may exacerbate barotrauma via impaired lung compliance. We hypothesize that ASM hyperreactivity and its sequelae are based on an early developmental lesion of ASM activity in hypoplastic lung. Sprague-Dawley rats were fed 100 mg nitrofen on Day 9.5 of pregnancy to induce lung hypoplasia in offspring (controls had vehicle alone). Normal and hypoplastic lung primordia were cultured from Day 13.5 of gestation at 37 degrees C in 5% CO(2) and loaded at 54 or 78 h with Ca(2+)-sensitive indicators: Fluo-4 for confocal imaging and Indo-1 or Fura-2 for photometric measurements of [Ca(2+)](i). Hypoplastic lung features spontaneous propagating ASM Ca(2+) transients with reduced frequency, increased amplitude, and significantly prolonged plateau duration, relative to control lung. Nonetheless, hypoplastic lung exhibits normal requirement for extracellular calcium entry and intracellular calcium release in initiation and regulation of ASM Ca(2+) waves. Early ASM dysfunction in lung hypoplasia is apparent as specific anomalies of Ca(2+) transients that indicate a problem with plasmalemmal ion channels/action potential generation. Elucidation of such an ASM lesion may allow pharmacologic amelioration not only of ASM hyperreactivity and its sequelae, but also of hypoplastic lung growth itself.

Spontaneous propagating calcium waves underpin airway peristalsis in embryonic rat lung.

Prenatal airways from diverse species exhibit spontaneous peristaltic contractions (airway peristalsis). These contractile waves appear coupled to and may function to regulate prenatal lung growth. They are unaffected by atropine or tetrodotoxin but abolished by nifedipine. Nevertheless, the mechanisms by which these contractile waves are generated, regulated, and propagated remain obscure. Using calcium imaging and whole embryonic lung organ culture, we demonstrate for the first time that peristalsis of the embryonic airway is driven by spontaneous, regenerative, temperature-sensitive calcium (Ca2+) waves. These Ca2+ waves propagate between individual airway smooth muscle cells coupled via gap junctions, are likely to be action potential-mediated, and are dependent on not only extracellular calcium entry via L-type voltage-gated channels but also intracellular Ca2+ stores. Thus, if airway peristalsis regulates lung growth, these findings mean that airway smooth muscle Ca2+ waves in turn regulate prenatal lung morphogenesis.

On the mechanisms whereby temperature affects excitation-contraction coupling in smooth muscle.

Moderate cooling of smooth muscle can modulate force production and may contribute to pathophysiological conditions, but the mechanisms underlying its effects are poorly understood. Interestingly, cooling increases force in rat ureter, but decreases it in guinea pigs. Therefore, this study used ureteric smooth muscle as a model system to elucidate the mechanisms of the effects of cooling on excitation-contraction coupling. Simultaneous recordings of force, intracellular [Ca(2+)], and electrical activity were made in intact ureter and ionic currents measured in isolated cells. The increase in force amplitude in rat ureter with cooling was found to be due to a significant increase in the duration of the Ca(2+) transient. This in turn was due to a marked prolongation of the action potential. In guinea pigs, both these parameters were much less affected by cooling. Examination of membrane currents revealed that differences in ion channel contribution to the action potential underlie these differences. In particular, cooling potentiated Ca(2+)-activated Cl(-) currents, which are present in rat but not guinea pig ureteric smooth muscle, and prolonged the plateau of the action potential and Ca(2+) entry. The force-Ca(2+) relationship revealed that the increased duration of the Ca(2+) transient was sufficient in the rat, but not in the guinea pig, to overcome kinetic lags produced in both species by cooling and potentiate force. Ca(2+) entry and release processes were largely temperature-insensitive, but the rate of relaxation was very temperature-sensitive. Effects of cooling on myosin light chain phosphatase, confirmed in experiments using calyculin A, appear to be the predominant mechanisms affecting relaxation. Thus, smooth muscle is diverse in its response to temperature, even when experimental variables, such as the mode of stimulation, are removed. Although the biochemical and mechanical events accompanying contraction are likely to be affected in similar ways by temperature, differences in electrical events lead to subsequent differences in these processes between smooth muscles.