Role of TRPC1 and TRPC3 channels in contraction and relaxation of mouse thoracic aorta.

BACKGROUND/AIMS: Canonical transient receptor potential (TRPC) channels modulate membrane potential and intracellular Ca(2+). We examined the role of TRPC1 and TRPC3 channels in vasocontraction and relaxation in mouse aorta. METHODS: Vasocontraction and relaxation of aorta from wild-type (WT), TRPC1 KO and TRPC3 knockout (KO) mice were measured for phenylephrine (Phe) and carbachol (CCh). Intracellular Ca(2+) was measured in primary aorta endothelial cells (EC) and whole cell K(+) current in freshly isolated smooth muscle cells (SMC). RESULTS AND CONCLUSION: TRPC1 KO aorta showed increased vasocontraction to Phe compared to WT and TRPC3 KO aorta due to diminished role of BK(Ca) channels. BK(Ca) mRNA (aorta) and whole cell current (SMC) were reduced versus WT. Contraction in WT aorta was increased to TRPC1 KO level by BK(Ca) channel inhibition. Relaxation to CCh was reduced in TRPC1 KO and TRPC3 KO aortas with concomitant reduction in EC Ca(2+) response. Pyr3 (TRPC3 blocker) reduced the Ca(2+) response to CCh in EC from WT, but not TRPC3 KO mice. In summary, TRPC1 attenuates receptor-mediated contraction through activation and/or expression of SMC BK(Ca) channels while TRPC3 does not contribute to receptor-mediated constriction. Both TRPC1 and TRPC3 participate in EC Ca(2+) influx and vasorelaxation of aorta.

Plasticity in intact A delta- and C-fibers contributes to cold hypersensitivity in neuropathic rats.

Cold hypersensitivity is a common sensory abnormality accompanying peripheral neuropathies and is difficult to treat. Progress has been made in understanding peripheral mechanisms underlying neuropathic pain but little is known concerning peripheral mechanisms of cold hypersensitivity. The aim of this study was to analyze the contribution of uninjured primary afferents to the cold hypersensitivity that develops in neuropathic rats. Rats with a lumbar 5 (L5) and L6 spinal nerve ligation (SNL, Chung model) but not sham, developed mechanical allodynia, evidenced by decreased paw withdrawal thresholds and increased magnitude of response to von Frey stimulation. Cold hypersensitivity also developed in SNL but not sham rats, evidenced by enhanced nociceptive behaviors induced by placement on a cold plate (6 degrees C) or application of icilin (a transient receptor potential M8 (TRPM8)/transient receptor potential A1 (TRPA1) receptor agonist) to nerve-injured hind paws. Single fiber recordings demonstrated that the mean conduction velocities of intact L4 cutaneous A delta- and C-fibers were not different between naive and SNL rats; however, mechanical thresholds of the A delta- but not the C-fibers were significantly decreased in SNL compared with naive. There was a higher prevalence of C-mechanoheat-cold (CMHC) fibers in SNL compared with naive, but the overall percentage of cold-sensitive C-fibers was not significantly increased compared with naive. This was in contrast to the numerous changes in A delta-fibers: the percentage of L4 cold sensitive A delta-, but not C-fibers, was significantly increased, the percentage of L4 icilin-sensitive A delta-, but not C-fibers, was significantly increased, the icilin-induced activity of L4 A delta-, but not C-fibers, was significantly increased. Icilin-induced activity was blocked by the TRPA1 antagonist Ruthenium Red. The results indicate plasticity in both A delta- and C-uninjured fibers, but A delta fibers appear to provide a major contribution to cold hypersensitivity in neuropathic rats.

Nongenomic actions of low concentration estrogens and xenoestrogens on multiple tissues.

Nongenomic estrogenic mechanisms offer an opportunity to explain the conundrum of environmental estrogen and plant estrogen effects on cells and animals at the very low concentrations which are prevalent in our environments and diets. Heretofore the actions of these compounds have not been adequately accounted for by laboratory tests utilizing assays for actions only via the genomic pathway of steroid action and the nuclear forms of estrogen receptor alpha and beta. Membrane versions of these receptors, and the newly described GPR30 (7TMER) receptor protein provide explanations for the more potent actions of xenoestrogens. The effects of estrogens on many tissues demand a comprehensive assessment of the receptors, receptor levels, and mechanisms that might be involved, to determine which of these estrogen mimetic compounds are harmful and which might even be used therapeutically, depending upon the life stage at which we are exposed to them.

P2X7 receptor stimulation of membrane internalization in a thyrocyte cell line.

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X(7) receptor activated a reversible, Ca(2+)-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C(m)) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5'-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C(m) without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2',3'-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2'4'-disulfonic acid, adenosine 5'-triphosphate-2'3'-dialdehyde, and Cu(2+). The capacitance decrease persisted in Na(+)-, Ca(2+)- and Cl(-)-free external saline or with Ca(2+)-free pipette solution. It is concluded that ATP activation of the inotropic P2X(7) receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca(2+) or influx of Na(+) or Ca(2+) through the receptor-gated channel.

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line.

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 microM Zn(2+), and 50 microM Cu(2+). Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC(50) for ATP stimulation of internalization was 440 microM in saline containing 2 mM Ca(2+) and 2 mM Mg(2+), and 33 microM in low-Mg(2+), nominally Ca(2+)-free saline. Overall, the results are most consistent with activation of a P2X(7) receptor by ATP(4-). However, low permeability to N-methyl-d-glucamine(+) and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X(7) receptor activation. ATP stimulation of internalization occurs in Na(+)-free, Ca(2+)-free, or low-Mg(2+) saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X(7) receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx.

Intracellular distribution and regulation of calpains in the thyroid gland.

We studied the distribution of calpains in various subcellular thyrocyte fractions and evaluated the possibility for direct activation of calpain localized in the plasma membrane by thyrotropin. Direct activation of calpain bound to the plasma membrane did not underlie transduction of the thyrotropin signal to Ca(2+)-dependent proteinases.