Cocaine- and amphetamine-regulated transcript peptide (CARTp): distribution and function in rat urinary bladder.

We investigated the distribution of CARTp(55-102) in rat lower urinary tract and evaluated its effect on urinary bladder function in vitro. Immunohistochemistry and a vertical isolated tissue bath system were used. Neurons, clusters of nonneuronal endocrine cells, and nerve fibers stained positive for CARTp(55-102) in young adult rat urinary bladder. The CARTp-expressing neuronal elements were nitric oxide synthase (NOS)- and tyrosine hydroxylase (TH)-IR, whereas all nonneuronal CARTp-IR elements stained positively only for TH (100 %). In isolated bladder strips, CARTp significantly increased the amplitude of electric field stimulation (EFS)-induced detrusor contractions at stimulation frequencies ≤12.5 Hz (p ≤ 0.001) as well as amplitude and frequency of spontaneous phasic urinary bladder smooth muscle (UBSM) contractions (p ≤ 0.05). The responses to CARTp stimulation were dose-dependent and increased in the presence of the urothelium. To determine if the CARTp increase in nerve-mediated contractions may involve an action of CARTp on specific neural pathways, we blocked cholinergic, purinergic, and adrenergic pathways and determined CARTp actions on EFS-medicated contractions. CARTp enhancement of EFS-mediated contractions does not involve alteration in purinergic, adrenergic, or cholinergic pathways. The study demonstrates that CARTp(55-102) is highly expressed in rat urinary bladder. CARTp increased the amplitude of EFS-induced detrusor contractions as well as the amplitude and frequency of spontaneous phasic urinary bladder smooth muscle contractions. We conclude that CARTp may alter the release of compounds from the urothelium that leads to an enhancement of UBSM contractility/excitability.

Low levels of K(ATP) channel activation decrease excitability and contractility of urinary bladder.

Activation of ATP-sensitive potassium (K(ATP)) channels can regulate smooth muscle function through membrane potential hyperpolarization. A critical issue in understanding the role of K(ATP) channels is the relationship between channel activation and the effect on tissue function. Here, we explored this relationship in urinary bladder smooth muscle (UBSM) from the detrusor by activating K(ATP) channels with the synthetic compounds N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZD-6169) and levcromakalim. The effects of ZD-6169 and levcromakalim on K(ATP) channel currents in isolated UBSM cells, on action potentials, and on related phasic contractions of isolated UBSM strips were examined. ZD-6169 and levcromakalim at 1.02 and 2.63 microM, respectively, caused half-maximal activation (K1/2) of K(ATP) currents in single UBSM cells (see Heppner TJ, Bonev A, Li JH, Kau ST, and Nelson MT. Pharmacology 53: 170-179, 1996). In contrast, much lower concentrations (K(1/2) = 47 nM for ZD-6169 and K1/2 = 38 nM for levcromakalim) caused inhibition of action potentials and phasic contractions of UBSM. The results suggest that activation of <1% of K(ATP) channels is sufficient to inhibit significantly action potentials and the related phasic contractions.

Voltage dependence of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary bladder smooth muscle.

Large-conductance Ca(2+)-dependent K(+) (BK(Ca)) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BK(Ca) currents and intracellular Ca(2+) revealed that BK(Ca) currents are activated by Ca(2+) release events (Ca(2+) sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca(2+) sparks and BK(Ca) currents and to determine the voltage dependence of the coupling of RyRs (Ca(2+) sparks) to BK(Ca) channels in UBSM. Ca(2+) sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca(2+) sparks caused BK(Ca) currents at all voltages tested, consistent with the BK(Ca) channels sensing approximately 10 microM Ca(2+). Membrane potential depolarization from -50 to -20 mV increased Ca(2+) spark and BK(Ca) current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca(2+) spark amplitude increasing by only 30% and BK(Ca) current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BK(Ca) current was quantitatively explained by the known voltage dependence of the Ca(2+) sensitivity of BK(Ca) channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca(2+) sensitivity of BK(Ca) channels, profoundly alters the coupling strength of Ca(2+) sparks to BK(Ca) channels.

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels.

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.

Involvement of L-type calcium channels in hypoxic relaxation of vascular smooth muscle.

In the systemic vasculature, hypoxia elicits a local vasodilator response that may be partially mediated by ionic channels on vascular smooth muscle, such as adenosine triphosphate sensitive K+ channels. Recent electrophysiological studies suggest that hypoxia may also inhibit voltage-dependent Ca2+ channels (L type) on peripheral vascular smooth muscle cells. We hypothesized that hypoxia elicits relaxation of vascular smooth muscle by inhibiting L-type Ca2+ channels. In endothelium-denuded rat thoracic aortic rings contracted with phenylephrine, mild and moderate hypoxia (PO2 35 and 20 mm Hg, respectively) elicited significant relaxation. Pretreatment with the L-type Ca2+ channel antagonist nifedipine completely inhibited mild hypoxic relaxation and diminished relaxation under moderate hypoxia, whereas glibenclamide, a blocker of adenosine triphosphate sensitive potassium channels, only attenuated the response to moderate hypoxia. In rings contracted with the L-type Ca2+ channel agonist (-)BAY K 8644 both mild and moderate hypoxia elicited almost complete relaxation. Furthermore, in rings contracted with hyperkalemic solutions (85 mM K+ or 120 mM K), mild and moderate hypoxia elicited significant relaxations. Thus, we conclude that hypoxia acts directly on vascular smooth muscle to cause relaxation in part by inhibiting L-type Ca2+ channels.

Maintained vasodilatory response to cromakalim after inhibition of nitric oxide synthesis.

Activation of vascular smooth-muscle adenosine triphosphate-sensitive potassium channels (KATP channels) causes membrane hyperpolarization, reduced entry of Ca2+ through L-type voltage-gated Ca2+ channels, and subsequent smooth-muscle relaxation. Conversely, opening of endothelial KATP channels elicits hyperpolarization but may induce Ca2+ influx and stimulation of endothelium-derived nitric oxide (EDNO) because these cells appear not to possess L-type Ca2+ channels. We therefore hypothesized that EDNO contributes to KATP channel-mediated vasodilation. To test this hypothesis, we examined vasodilatory responses to the KATP channel opener cromakalim in conscious rats, perfused rat tail artery segments, and isolated perfused rat lungs in the presence or absence of the EDNO synthesis inhibitor Nomega-nitro-L-arginine (L-NNA). Additionally, we compared the effect of cromakalim with the EDNO-dependent dilator bradykinin on NO production and intracellular Ca2+ in cultured rat pulmonary artery endothelial cells. Vasodilatory profiles to cromakalim were unaffected by L-NNA in conscious rats, tail arteries, and isolated lungs. Consistent with these results, cromakalim had no apparent effect on either NO synthesis or Ca2+ levels in cultured endothelial cells. These data suggest a lack of a role for EDNO in contributing to KATP-channel-mediated vasodilation in the rat.

Glibenclamide does not reverse attenuated vasoreactivity to acute or chronic hypoxia.

Recent studies from our laboratory have shown that acute and chronic hypoxic exposures are associated with attenuated systemic vasoreactivity in conscious rats. The present studies examined the role of adenosine triphosphate-sensitive potassium channels (KATP channels) in modulating the pressor and vasoconstrictor responses to phenylephrine (PE) in conscious instrumented rats 1) during acute hypoxia or 2) after chronic hypoxic exposure. Mean arterial pressure, mean cardiac output, and total peripheral resistance were assessed before and after graded infusions of PE in both groups of rats under normoxic or hypoxic conditions. Additionally, the role of KATP channels in attenuating vasoreactivity was determined by administration of glibenclamide (KATP channel blocker) before PE infusions. Acute hypoxia (12% O2) was associated with reduced pressor and constrictor responses to PE in control animals. Furthermore, acute return to room air did not restore the pressor and constrictor responses in the chronically hypoxic rats. Glibenclamide infusion did not influence the pressor or vasoconstrictor responses to PE in either group of animals during normoxia or acute hypoxia. Therefore, our data suggest that opening of KATP channels is not involved in the attenuated vasoreactivity associated with acute and chronic hypoxia in the conscious rat.