We examined the contribution of large-conductance, Ca²⁺-sensitive K⁺ (MaxiK) channel to β₂-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between β₂-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through β₂-type of adrenoceptor since the response was antagonized in a competitive fashion by a β₂-selective adrenoceptor antagonist butoxamine with a pA₂ value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 μM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 μM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 μM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 μg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein G_s. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates β₂-adrenoceptor-activated relaxation; 2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between β₂-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by G_s operates in cAMP-independent coupling between β₂-adrenoceptor and this ion channel.

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It has recently been suggested that RhoA plays an important role in the enhancement of Ca²⁺ sensitization observed in smooth muscle contraction. In the present study, the expression of rhoA mRNA in the bronchial smooth muscle of antigen-induced airway hyperresponsive rats was compared with that of control animals. Reverse transcription-polymerase chain reaction experiments using total RNA from these tissue specimens and the specific primers revealed rhoA mRNA to be expressed in bronchial smooth muscle of the rat. The rhoA mRNA expression in bronchial smooth muscle of the hyperresponsive rats was significantly increased in comparison to that of control animals. It is thus possible that upregulation of RhoA protein might be involved in the mechanism underlying the increased contractility of the bronchial smooth muscle which occurs with airway hyperresponsiveness.

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Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptors were present in circular smooth muscle cells of the guinea-pig gastric antrum. Nonselective cation channels (NSCC) activated by ACh or CCh are coupled to pertussis toxin (PTX)-sensitive Go protein through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach. CCh-activated currents (I_CCh), especially the steady-state I-V relationship of I_CCh showed a chracteristic U-shaped curve; reversal potential of around 0 mV and inward rectification at around +15 mV and a negative slope conductance at negative potential range. Under physiological conditions, the measured single channel conductance of NSCC was approximately 25 pS. The single channel conductance was modulated by external monovalent and divalent cations including Na⁺, Cs⁺, Li⁺, and Ca²⁺ through changing both the open probability and unitary conductance. Through the NSCC, Ca²⁺ can move into the cell from extracellular solution as well as Na⁺. Calculated fractional Ca²⁺ current of I_CCh (f_Ca) was around 1% at the 2 mM [Ca²⁺]_o and at the 4 mM [Ca²⁺]_o, f_Ca was 2.3%. Quinidine blocked I_CCh potently in a reversible manner; IC₅₀ was 0.25 μM. There were two kinds of I_CCh modulations through Ca²⁺-dependent pathways in guinea-pig gastric smooth muscle cells; 1) Facilitation of I_CCh via Ca²⁺/CaM-dependent MLCK pathway, 2) Desensitization of I_CCh via Ca²⁺-dependent PKC pathway. In the mouse stomach, all seven types of TRPC mRNA were detected with RT-PCR. On the basis of electrophysiological, pharmacological, and molecular biological experiments, we reported the mTRPC5 as a candidate for the NSCC activated by muscarinic stimulation in mouse stomach.

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In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor - endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K⁺), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

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