Airway smooth muscle relaxation results from a reduction in the frequency of Ca(2+ )oscillations induced by a cAMP-mediated inhibition of the IP(3 )receptor

BACKGROUND: It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca(2+ )oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca(2+ )oscillations. METHODS: The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca(2+ )signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy. RESULTS: All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca(2+ )oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca(2+ )oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca(2+ )oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca(2+ )stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca(2+ )available to stimulate contraction because an elevation of intracellular Ca(2+ )concentration induced by exposure to a Ca(2+ )ionophore (ionomycin) or by photolysis of caged-Ca(2+ )did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca(2+)-activated K(+ )channels, which would be expected to increase Ca(2+ )influx and contraction. By contrast, the photolysis of caged-IP(3 )in the presence of agonist, to further elevate the intracellular IP(3 )concentration, reversed the slowing of the frequency of the Ca(2+ )oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP(3)R to IP(3 )was reduced by FSK and this was supported by the reduced ability of IP(3 )to release Ca(2+ )in SMCs in the presence of FSK. CONCLUSION: These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca(2+ )oscillation frequency by reducing internal Ca(2+ )release through IP(3 )receptors

Signal transduction underlying the control of urinary bladder smooth muscle tone by muscarinic receptors and β-adrenoceptors

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M3 subtype, with the M2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by β-adrenoceptors, in most species involving a strong β3-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of β-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to β-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in β-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and β-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder

Airway smooth muscle as a target of asthma therapy: history and new directions

Ultimately, asthma is a disease characterized by constriction of airway smooth muscle (ASM). The earliest approach to the treatment of asthma comprised the use of xanthines and anti-cholinergics with the later introduction of anti-histamines and anti-leukotrienes. Agents directed at ion channels on the smooth muscle membrane (Ca(2+ )channel blockers, K(+ )channel openers) have been tried and found to be ineffective. Functional antagonists, which modulate intracellular signalling pathways within the smooth muscle (β-agonists and phosphodiesterase inhibitors), have been used for decades with success, but are not universally effective and patients continue to suffer with exacerbations of asthma using these drugs. During the past several decades, research energies have been directed into developing therapies to treat airway inflammation, but there have been no substantial advances in asthma therapies targeting the ASM. In this manuscript, excitation-contraction coupling in ASM is addressed, highlighting the current treatment of asthma while proposing several new directions that may prove helpful in the management of this disease

Insulin attenuates agonist-evoked calcium transients in vascular smooth muscle cells

Insulin may decrease the contractile response of vascular smooth muscle to vasoactive agents. This could be due to interactions of insulin with the effects of vasoactive agonists on intracellular free calcium transients in vascular smooth muscle cells. This study assesses the effects of physiological doses of insulin (70 microU/mL) on calcium responses in cultured vascular smooth muscle cells (primary unpassaged and passaged) to angiotensin II (1 nmol/L), arginine vasopressin (10 nmol/L), and norepinephrine (10 mumol/L). Intracellular free Ca2+ concentrations in single cells were measured microphotometrically using fura 2-AM. Insulin, angiotensin II, arginine vasopressin, and norepinephrine significantly increased calcium (to 115 +/- 7, 183 +/- 20, 184 +/- 15, and 168 +/- 12 nmol/L, respectively, from basal calcium of 90 +/- 10 nmol/L). Insulin significantly attenuated the agonist-induced calcium responses. The effects of insulin were almost completely inhibited by diltiazem, staurosporine, calphostin C, and thapsigargin. In conclusion, insulin stimulates calcium transients but blunts agonist-mediated calcium rises in vascular smooth muscle cells. These responses are related to regulatory effects of insulin on cellular calcium homeostasis and may explain how insulin modulates vascular smooth muscle contraction

Blunted attenuation of angiotensin II-mediated Ca²⁺ transients by insulin in cultured unpassaged vascular smooth muscle cells from spontaneously hypertensive rats

Insulin attenuates vasoconstrictor-stimulated intracellular calcium ([Ca2+]i) responses in cells from normotensive rats. To determine whether these effects may be altered in hypertension, this study assesses the effects of physiologic concentrations of insulin on angiotensin II-stimulated [Ca2+]i in primary unpassaged cultured vascular smooth muscle cells (VSMC) from mesenteric arteries from spontaneously hypertensive rats (SHR), Wistar Kyoto (WKY), and Wistar rats. [Ca2+]i was measured microphotometrically in cells from 3-, 9-, and 17-week-old rats by the Fura 2 methodology. Basal, angiotensin II-stimulated (1 nmol/L), and insulin-attenuated (70 microU/mL) angiotensin II-induced [Ca2+]i did not differ in VSMC obtained from 3-week-old WKY, Wistar, and SHR. Basal and angiotensin II-stimulated [Ca2+]i in VSMC from 9- and 17-week-old SHR was significantly greater (P < .01) than that in cells from age-matched WKY and Wistar rats. Insulin decreased angiotensin II-stimulated [Ca2+]i responses in all groups, but the effect was significantly blunted in cells from 9- and 17-week-old SHR. The magnitude of inhibition of angiotensin II-stimulated [Ca2+]i responses induced by insulin was 63 +/- 12 nmol/l (WKY), 60 +/- 10 nmol/L (Wistar), and 28 +/- 8 nmol/L (SHR), (P < .01 v normotensive) in cells from 9-week-old rats and 70 +/- 15 nmol/L (WKY), 67 +/- 12 nmol/L (Wistar), and 25 +/- 10 nmol/L (SHR) (P < .01 v normotensive) in cells from 17-week-old rats. Insulin increased angiotensin II-stimulated [Ca2+]i recovery rate to basal values in cells from WKY rats, but had no effect on the rate of recovery in VSMC from SHR

Mesenteric vascular smooth muscle cells from SHR display increased Ca²⁺ responses to angiotensin II but not to endothelin-1

<p>Objective: To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats.</p> <p>Methods: Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology.</p> <p>Results: Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups.</p> <p>Conclusions: In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Angll are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.</p&gt