Kirk W. Evanson

LRRC26 is a Functional BK Channel Auxiliary γ Subunit in Arterial Smooth Muscle Cells

Rationale: Smooth muscle cell (myocyte) large-conductance calcium (Ca)2+-activated potassium (BK) channels are functionally significant modulators of arterial contractility. Arterial myocytes express both pore-forming BK&agr; and auxiliary &bgr;1 subunits, which increase channel Ca2+ sensitivity. Recently, several leucine-rich repeat containing (LRRC) proteins have been identified as auxiliary &ggr; subunits that elevate the voltage sensitivity of recombinant and prostate adenocarcinoma BK channels. LRRC expression and physiological functions in native cell types are unclear. Objective: Investigate the expression and physiological functions of leucine-rich repeat containing protein 26 (LRRC26) in arterial myocytes. Methods and Results: Reverse transcription polymerase chain reaction and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence resonance energy transfer microscopy, and coimmunoprecipitation indicated that LRRC26 was located in close spatial proximity to, and associated with, plasma membrane BK&agr; subunits. LRRC26 knockdown (RNAi) reduced total and surface LRRC26, but did not alter BK&agr; or &bgr;1, proteins in arteries. LRRC26 knockdown did not alter Ca2+ sparks but reduced BK channel voltage sensitivity, which decreased channel apparent Ca2+ sensitivity and transient BK current frequency and amplitude in myocytes. LRRC26 knockdown also increased myogenic tone over a range (40–100 mm Hg) of intravascular pressures, and reduced vasoconstriction to iberiotoxin and vasodilation to NS1619, BK channel inhibitors and activators, respectively. In contrast, LRRC26 knockdown did not alter depolarization (60 mmol/L K+)–induced vasoconstriction. Conclusions: LRRC26 is expressed, associates with BK&agr; subunits, and elevates channel voltage- and apparent Ca2+ sensitivity in arterial myocytes to induce vasodilation. This study indicates that arterial myocytes express a functional BK channel &ggr; subunit.

The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator

•  Voltage‐dependent L‐type Ca2+ (CaV1.2) channels are the major Ca2+ influx pathway and are central to contractility regulation in arterial smooth muscle cells. •  CaV1.2 exists as a full‐length channel and undergoes cleavage to a short CaV1.2 and a C‐terminus (CCt) fragment in rat and human arterial smooth muscle cells. •  CCt decreases CaV1.2 transcription and shifts the voltage dependence of current activation and inactivation to more depolarized potentials in arterial smooth muscle cells. •  CCt reduces pressure‐ and depolarization‐induced vasoconstriction. •  CCt is a bi‐modal vasodilator.

ATP overflow in skeletal muscle 1A arterioles

The purpose of this study was to investigate the sources of ATP in the 1A arteriole, and to investigate age‐related changes in ATP overflow. Arterioles (1A) from the red portion of the gastrocnemius muscle were isolated, cannulated and pressurized in a microvessel chamber with field stimulation electrodes. ATP overflow was determined using probes specific for ATP and null probes that were constructed similar to the ATP probes, but did not contain the enzyme coating. ATP concentrations were determined using a normal curve (0.78 to 25 μmol l−1 ATP). ATP overflow occurred in two phases. Phase one began in the first 20 s following stimulation and phase two started 35 s after field stimulation. Tetrodotoxin, a potent neurotoxin that blocks action potential generation in nerves, abolished both phases of ATP overflow. α1‐Receptor blockade resulted in a small decrease in ATP overflow in phase two, but endothelial removal resulted in an increase in ATP overflow. ATP overflow was lowest in 6‐month‐old rats and highest in 12‐ and 2‐month‐old rats (P < 0.05). ATP overflow measured via biosensors was of neural origin with a small contribution from the vascular smooth muscle. The endothelium seems to play an important role in attenuating ATP overflow in 1A arterioles.

Neuropeptide Y overflow and metabolism in skeletal muscle arterioles

Non‐technical summary  Neuropeptide Y (NPY) is involved in a number of vascular physiological processes that affect sympathetic neurotransmission and angiogenesis. While NPY is of physiological significance, very little is known regarding local overflow characteristics at specific levels of the vasculature. Through the use of a new technique, we were able to quantify NPY overflow from isolated skeletal muscle arterioles of female rats. We observed age‐related differences in NPY overflow and its degradation via dipeptidyl peptidase IV. These results will provide insights into the release and breakdown of NPY at local levels of the vasculature.

Influence of estradiol supplementation on neuropeptide Y neurotransmission in skeletal muscle arterioles of F344 rats

The effects of estradiol on neuropeptide Y (NPY) neurotransmission in skeletal muscle resistance vessels have not been described. The purpose of this study was to determine the effects of long-term estradiol supplementation on NPY overflow, degradation, and vasoconstriction in gastrocnemius first-order arterioles of adult female rats. Female rats (4 mo; n = 34) were ovariectomized (OVX) with a subset (n = 17) receiving an estradiol pellet (OVE; 17β-estradiol, 4 μg/day). After conclusion of the treatment phase (8 wk), arterioles were excised, placed in a physiological saline solution (PSS) bath, and cannulated with micropipettes connected to albumin reservoirs. NPY-mediated vasoconstriction via a Y(1)-agonist [Leu31Pro34]NPY decreased vessel diameter 44.54 ± 3.95% compared with baseline; however, there were no group differences in EC(50) (OVE: -8.75 ± 0.18; OVX: -8.63 ± 0.10 log M [Leu31Pro34]NPY) or slope (OVE: -1.11 ± 0.25; OVX: -1.65 ± 0.34% baseline/log M [Leu31Pro34]NPY). NPY did not potentiate norepinephrine-mediated vasoconstriction. NPY overflow experienced a slight increase following field stimulation and significantly increased (P < 0.05) over control conditions in the presence of a DPPIV inhibitor (diprotin A). Estradiol status did not affect DPPIV activity. These data suggest that NPY can induce a moderate decrease in vessel diameter in skeletal muscle first-order arterioles, and DPPIV is active in mitigating NPY overflow in young adult female rats. Long-term estradiol supplementation did not influence NPY vasoconstriction, overflow, or its enzymatic breakdown in skeletal muscle first-order arterioles.

Arterial smooth muscle cell PKD2 (TRPP1) channels control systemic blood pressure

Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-receptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. In summary, we show for the first time that arterial myocyte PKD2 channels control systemic blood pressure and targeting reduces high blood pressure.

Elevated plasma catecholamines functionally compensate for the reduced myogenic tone in smooth muscle STIM1 knockout mice but with deleterious cardiac effects

Aims Stromal interaction molecule 1 (STIM1) has emerged as an important player in the regulation of growth and proliferation of smooth muscle cells. Therefore, we hypothesized that STIM1 plays a crucial role in the maintenance of vascular integrity. The objective of this study was to evaluate whether reduced expression of STIM1 could modify the structure and function of the vasculature, leading to changes in blood pressure (BP). Methods and results Smooth muscle-specific STIM1 knockout (sm-STIM1 KO) in mice resulted in arteries with ∼80% reduced STIM1 protein expression as compared with control mice. Mesenteric vessels exposed to increasing transmural pressure revealed attenuated myogenic reactivity and reduced vasoconstrictor response to phenylephrine in sm-STIM1 KO arteries. BP monitored via telemetry in sm-STIM1 KO and matched controls did not reveal differences. However, heart rate was significantly increased in sm-STIM1 KO mice. Consistent with these findings, plasma catecholamine levels were higher in sm-STIM1 KO than in control mice. Increased sympathetic activity in sm-STIM1 KO mice was unmasked by apha1-adrenergic receptor inhibitor (prazosin) and by treatment with the ganglion-blocking agent, hexamethonium. Both treatments resulted in a greater reduction of BP in sm-STIM1 KO mice. Cytoskeleton of cultured smooth muscle cells was studied by immunocytochemistry using specific antibodies. Staining for actin and vinculin revealed significant alterations in the cytoskeletal architecture of cells isolated from sm-STIM1 KO arteries. Finally, although sm-STIM1 KO mice were protected from Ang II-induced hypertension, such treatment resulted in significant fibrosis and a rapid deterioration of cardiac function. Conclusions STIM1 deletion in smooth muscle results in attenuated myogenic tone and cytoskeletal defects with detrimental effects on the mechanical properties of arterial tissue. Although BP is maintained by elevated circulating catecholamine, this compensatory stimulation has a deleterious long-term effect on the myocardium.

ATP metabolism in skeletal muscle arterioles

The purpose of this study was to investigate the metabolism of Adenosine triphosphate (ATP) in skeletal muscle resistance arterioles and to determine whether this metabolism is altered during the rapid growth phase of the rat. We attempted to quantify ATP metabolism in gastrocnemius first‐order arterioles from 8‐, 10‐, and 12‐week‐old rats. We measured ATP metabolism using an ATPase/GTPase assay with whole vessel segments as well as using a real‐time adenosine biosensor following electric field stimulation. Our first method of measuring ATP metabolism allowed us to measure the amount of free phosphate produced with ATP as a substrate. When ecto‐nucleotidase activity was inhibited by ARL67156, pyridoxal phosphate‐6‐azophenly‐2′, 4′‐disulfonic acid (PPADS), or suramin prior to adding ATP, we found that the rate of phosphate production was significantly reduced by 27%, 21%, and 22%, respectively (P < 0.05). Our second method of measuring ATP metabolism allowed us to measure the amount of adenosine produced following electric field stimulation of the arteriole with and without nucleotidase inhibitors. Surprisingly, we found that adenosine overflow was not attenuated by nucleotidase inhibitors. We concluded that ecto‐phosphodieterase/phyrophophatase (E‐NPP), ecto‐diadenosine polyphosphatase (ApnA), NTPDase1 and 2, and E5NT may be present on the gastrocnemius 1A arteriole and do play a role in ATP metabolism. Between the ages of 8 weeks and 12 weeks, however, overall ATP metabolism may not change.