cGMP-dependent protein kinase (PKG) mediates the anticontractile capacity of perivascular adipose tissue.

AIM: The aim of this study was to investigate the role of cGMP-dependent protein kinase (PKG) in mediating the anticontractile function of perivascular adipose tissue (PVAT) and whether its activation can rescue PVAT activity which is lost in an experimental model of inflammation. METHODS AND RESULTS: Contractile responses to norepinephrine were assessed using wire myography from small arterial segments obtained from PKG(-/-), PKG(+/+), adipo(-/-), and C57Bl6/J mice with and without PVAT during normal oxygenation and hypoxia. An anticontractile effect of PVAT was observed in control blood vessels. This was not present in arteries from PKG(-/-) or PKG(+/+) with inhibition of PKG signalling using DT-2/ODQ. Hypoxia-induced loss of PVAT function was rescued by ANP activation of PKG as there was no effect in blood vessels from PKG(-/-) mice or in the presence of DT-2. Solution transfer studies demonstrated that PKG was necessary for the normal paracrine effects of PVAT on smooth muscle and endothelium. PKG activation by atrial natriuretic peptide (ANP) did not restore the absent PVAT anticontractile capacity in arteries from adiponectin(-/-) mice; however, inhibition of PKG did not further abrogate this effect suggesting dysregulation of PKG signalling pathways in this model. The absence of PKG was associated with reduced adipocyte adiponectin expression. CONCLUSION: PKG plays a key role in regulating normal PVAT function both in modulating anticontractile factor release from adipocytes as well as being essential for its downstream dilator function in arterial smooth muscle.

Perivascular adipose tissue-derived adiponectin activates BK(Ca) channels to induce anticontractile responses.

This study aims to identify the potential mechanisms by which perivascular adipose tissue (PVAT) reduces tone in small arteries. Small mesenteric arteries from wild-type and large-conductance Ca(2+)-activated K(+) (BKCa) channel knockout mice were mounted on a wire myograph in the presence and absence of PVAT, and contractile responses to norepinephrine were assessed. Electrophysiology studies were performed in isolated vessels to measure changes in membrane potential produced by adiponectin. Contractile responses from wild-type mouse small arteries were significantly reduced in the presence of PVAT. This was not observed in the presence of a BKCa channel inhibitor or with nitric oxide synthase (NOS) inhibition or in BKCa or adiponectin knockout mice. Solution transfer experiments demonstrated the presence of an anticontractile factor released from PVAT. Adiponectin-induced vasorelaxation and hyperpolarization in wild-type arteries were not evident in the absence of or after inhibition of BKCa channels. PVAT from BKCa or adiponectin knockout mice failed to elicit an anticontractile response in wild-type arteries. PVAT releases adiponectin, which is an anticontractile factor. Its effect on vascular tone is mediated by activation of BKCa channels on vascular smooth muscle cells and adipocytes and by endothelial mechanisms.

Amplified NO/cGMP-mediated relaxation and ryanodine receptor-to-BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice.

BACKGROUND AND PURPOSE: Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK(Ca) ) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK(Ca) activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+) release from the SR via ryanodine receptors (RyRs) is important for BK(Ca) channel activation in response to NO/cGMP. EXPERIMENTAL APPROACH: In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB(-/-)) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS). KEY RESULTS: In CCSM strips from PLB(-/-) mice, a model of increased SR Ca(2+) load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB(-/-) strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB(-/-) , as demonstrated by a shift in the concentration-response curve towards lower concentrations. Blocking RyRs and BK(Ca) channels diminished the induced relaxations and eliminated the difference between wild-type and PLB(-/-). CONCLUSIONS AND IMPLICATIONS: NO/cGMP activates BK(Ca) channels through RyR-mediated Ca(2+) release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca(2+) concentrations.

Calcium signaling in smooth muscle.

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

Nerve-evoked purinergic signalling suppresses action potentials, Ca2+ flashes and contractility evoked by muscarinic receptor activation in mouse urinary bladder smooth muscle.

Contraction of urinary bladder smooth muscle (UBSM) is caused by the release of ATP and ACh from parasympathetic nerves. Although both purinergic and muscarinic pathways are important to contraction, their relative contributions and signalling mechanisms are not well understood. Here, the contributions of each pathway to urinary bladder contraction and the underlying electrical and Ca(2+) signalling events were examined in UBSM strips from wild type mice and mice deficient in P2X1 receptors (P2X1(-/-)) before and after pharmacological inhibition of purinergic and muscarinic receptors. Electrical field stimulation was used to excite parasympathetic nerves to increase action potentials, Ca(2+) flash frequency, and force. Loss of P2X1 function not only eliminated action potentials and Ca(2+) flashes during stimulation, but it also led to a significant increase in Ca(2+) flashes following stimulation and a corresponding increase in the force transient. Block of muscarinic receptors did not affect action potentials or Ca(2+) flashes during stimulation, but prevented them following stimulation. These findings indicate that nerve excitation leads to rapid engagement of smooth muscle P2X1 receptors to increase action potentials (Ca(2+) flashes) during stimulation, and a delayed increase in excitability in response to muscarinic receptor activation. Together, purinergic and muscarinic stimulation shape the time course of force transients. Furthermore, this study reveals a novel inhibitory effect of P2X1 receptor activation on subsequent increases in muscarinic-driven excitability and force generation.

Hypercontractility and impaired sildenafil relaxations in the BKCa channel deletion model of erectile dysfunction.

Erectile dysfunction (ED) can be elicited by a variety of pathogenic factors, particularly impaired formation of and responsiveness to nitric oxide (NO) and the downstream effectors soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI). One important target of PKGI in smooth muscle is the large-conductance, Ca2+ -activated potassium (BKCa) channel. In our previous report (42), we demonstrated that deletion of the BKCa channel in mice induced force oscillations and led to reduced nerve-evoked relaxations and ED. In the current study, we used this ED model to explore the role of the BKCa channel in the NO/sGC/PKGI pathway. Electrical field stimulation (EFS)-induced contractions of corpus cavernosum smooth muscle strips were significantly enhanced in the absence of BKCa channel function. In strips precontracted with phenylephrine, EFS-induced relaxations were converted to contractions by inhibition of sGC, and this was further enhanced by loss of BK channel function. Sildenafil-induced relaxations were decreased to a similar extent by inhibition of sGC or BKCa channels. At concentrations >1 microM, sildenafil caused relaxations independent of inhibition of sGC or BKCa channels. Sildenafil did not affect the enhanced force oscillations that were induced by the loss of BKCa channel function. Yet, these oscillations could be completely eliminated by blocking L-type voltage-dependent Ca2+ channels (VDCCs). These results suggest that therapeutically relevant concentrations of sildenafil act through cGMP and BKCa channels, and loss of BKCa channel function leads to hypercontractility, which depends on VDCCs and cannot be modified by the cGMP pathway.

Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels.

In the urinary bladder, contractions of the detrusor muscle and urine voiding are induced by the neurotransmitters ACh and ATP, released from parasympathetic nerves. Activation of K(+) channels, in particular the large-conductance Ca(2+)-activated K(+) (BK) channels, opposes increases in excitability and contractility of urinary bladder smooth muscle (UBSM). We have shown that deleting the gene mSlo1 in mice (Slo(-/-)), encoding the BK channel, leads to enhanced nerve-mediated and neurotransmitter-dependent contractility of UBSM (38). Here, we examine the location of the BK channel in urinary bladder strips from mouse. Immunohistochemical analysis revealed that the channel is expressed in UBSM but not in nerves that innervate the smooth muscle. The relationship between electrical field stimulation and force generation of the cholinergic and purinergic pathways was examined by applying blockers of the respective receptors in UBSM strips from wild-type and from Slo(-/-) (knockout) mice. In wild-type strips, the stimulation frequency required to obtain a half-maximal force was significantly lower for the purinergic (7.2 +/- 0.3 Hz) than the cholinergic pathway (19.1 +/- 1.5 Hz), whereas the maximum force was similar. Blocking BK channels with iberiotoxin or ablation of the Slo gene increased cholinergic- and purinergic-mediated force at low frequencies, i.e., significantly decreased the frequency for a half-maximal force. Our results indicate that the BK channel has a very significant role in reducing both cholinergic- and purinergic-induced contractility and suggest that alterations in BK channel expression or function could contribute to pathologies such as overactive detrusor.

Calcium-activated potassium channels and the regulation of vascular tone.

Different calcium signals in the endothelium and smooth muscle target different types of Ca2+-sensitive K+ channels to modulate vascular function. These differential calcium signals and targets represent multilayered opportunities for prevention and/or treatment of vascular dysfunctions.

Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel.

Penile erection is dependent on the nitric oxide (NO)/cGMP-dependent protein kinase I (PKGI) pathway. One important target of PKGI in smooth muscle is the large-conductance, calcium-activated potassium (BK) channel, which upon activation hyperpolarizes the smooth muscle cell membrane, causing relaxation. Relaxation of arterial and corpus cavernosum smooth muscle (CCSM) is necessary to increase blood flow into the corpora cavernosa that leads to penile tumescence. We investigated the functional role of BK channels in the corpus cavernosum utilizing a knock-out mouse lacking the Slo gene (Slo-/-) responsible for the pore-forming subunit of the BK channel. Whole-cell currents were recorded from isolated CCSM cells of Slo+/+ and Slo-/- mice. Iberiotoxin-sensitive voltage- and [Ca2+]-activated K+ currents, the latter activated by local transient calcium releases (calcium sparks), were present in Slo+/+ CCSM cells, but absent in Slo-/- cells. CCSM strips from Slo-/- mice demonstrated a four-fold increase in phasic contractions, in the presence of phenylephrine. Nerve-evoked relaxations of precontracted strips were reduced by 50%, both in strips from Slo-/- mice and by blocking BK channels with iberiotoxin in the Slo+/+ strips. Consistent with the in vitro results, in vivo intracavernous pressure exhibited pronounced oscillations in Slo-/- mice, but not in Slo+/+ mice. Furthermore, intracavernous pressure increases to nerve stimulation, in vivo, were reduced by 22% in Slo-/- mice. These results indicate that the BK channel has an important role in erectile function, and loss of the BK channel leads to erectile dysfunction.

Overactive bladder and incontinence in the absence of the BK large conductance Ca2+-activated K+ channel.

BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo(-/-) mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo(-/-) mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients ("calcium sparks") and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo(-/-) mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence.