Biomedical subjects

Leanne L Cribbs

Publications and source records attributed to Leanne L Cribbs.

8 recordsLinked to original sources

PubMed2006

Protein kinase Cepsilon-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes.

The myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a cytoskeletal protein implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. However, its precise role in cardiomyocyte growth, and its PKC-dependent regulation have not been fully explored. In this report, we show that MARCKS is expressed and phosphorylated under basal conditions in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively). The PKC activators phenylephrine, angiotensin II, and endothelin-1 (ET) further increased MARCKS phosphorylation, with ET inducing the greatest response. To determine which PKC isoenzyme was responsible for agonist-induced MARCKS phosphorylation, NRVM and ARVM were infected with replication-defective adenoviruses (Adv) encoding wildtype (wt) and constitutively active (ca) mutants of PKCepsilon, PKCdelta, and PKCalpha. Only PKCepsilon increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKCepsilon reduced basal and ET-stimulated pMARCKS. dnPKCepsilon overexpression also prevented ET-induced, apparent co-localization of pMARCKS with f-actin staining structures. Adv-mediated overexpression of GFP-tagged, wtMARCKS (wtMARCKS-GFP) increased phosphorylation of focal adhesion kinase (FAK) and also increased NRVM surface area. In contrast, overexpression of a GFP-tagged, non-phosphorylatable (np) MARCKS mutant (npMARCKS-GFP) decreased basal and ET-induced endogenous MARCKS and FAK phosphorylation, and blocked the ET-induced increase in NRVM surface area. We conclude that MARCKS is expressed in cardiomyocytes, is phosphorylated by PKCepsilon, and participates in the regulation of FAK phosphorylation and cell spreading.

Adenoviridae↗

PubMed2006

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells.

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.

Action Potentials↗

PubMed2006

T-type Ca2+ channels in vascular smooth muscle: multiple functions.

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.

Animals↗

PubMed2006

Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca(2+) entry in vascular smooth muscle cells.

Capacitative Ca(2+) entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) have demonstrated that depletion of intracellular Ca(2+) stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I(SOC))]. Because of different recording conditions, it has not been previously determined whether I(SOC) corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I(SOC), including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd(3+), were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated I(SOC) was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I(SOC) was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I(SOC)/CCE pathway.

Animals↗

PubMed2005

Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells.

In this study we investigated the T-type calcium channel and its involvement in the cell division of U87MG cultured glioma cells and N1E-115 neuroblastoma cells. Using Western blot analysis, we found that expression of both alpha1G and alpha1H subunits of the T-type calcium channel decreased during conditions associated with a decrease in proliferation as evidenced by increased expression of cyclin D1, a marker for non-proliferating cells. Both serum starvation and application of mibefradil, a selective T-type calcium channel antagonist, resulted in a 50% decrease in the expression of alpha1G and alpha1H and a 700-900% increase in levels of cyclin D1 in U87MG and N1E-115 cells, respectively. Furthermore, overexpression of the alpha1H subunit resulted in a two-fold increase in cell proliferation compared to control cultures or cultures receiving an empty vector. In contrast, blocking expression of the alpha1G subunit using antisense oligonucleotides lead to a 70% decrease in proliferation of U87MG and N1E-115 cells compared to control cultures or cultures receiving a scrambled oligonucleotide. Our findings suggest that proliferation of U87MG glioma cells and N1E-115 is regulated by T-type calcium channel expression.

Animals↗

PubMed2004

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking.

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.

Adenoviridae↗

PubMed2003

Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels.

Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.

Acetylcholine↗

PubMed2002

Single-channel pharmacology of mibefradil in human native T-type and recombinant Ca(v)3.2 calcium channels.

To study the molecular pharmacology of low-voltage-activated calcium channels in biophysical detail, human medullary thyroid carcinoma (hMTC) cells were investigated using the single-channel technique. These cells had been reported to express T-type whole-cell currents and a Ca(v)3.2 (or alpha 1H) channel subunit. We observed two types of single-channel activity that were easily distinguished based on single-channel conductance, voltage dependence of activation, time course of inactivation, rapid gating kinetics, and the response to the calcium agonist (S)-Bay K 8644. Type II channels had biophysical properties (activation, inactivation, conductance) typical for high-voltage-activated calcium channels. They were markedly stimulated by 1 microM (S)-Bay K 8644, allowing to identify them as L-type channels. The channel termed type I is a low-voltage-activated, small-conductance (7.2 pS) channel that inactivates rapidly and is not modulated by (S)-Bay K 8644. Type I channels are therefore classified as T-type channels. They were strongly inhibited by 10 microM mibefradil. Mibefradil block was caused by changes in two gating parameters: a pronounced reduction in fraction of active sweeps and a slight shortening of the open-state duration. Single recombinant low-voltage-activated T-type calcium channels were studied in comparison, using human embryonic kidney 293 cells overexpressing the pore-forming Ca(v)3.2 subunit. Along all criteria examined (mechanisms of block, extent of block), recombinant Ca(v)3.2 interact with mibefradil in the same way as their native counterparts expressed in hMTC cells. In conclusion, the pharmacologic phenotype of these native human T-type channels--as probed by mibefradil--is similar to recombinant human Ca(v)3.2.

Calcium Channel Blockers↗