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Cadmium (Cd) is frequently used in various industrial applications and is a ubiquitous environmental toxicant, also present in tobacco smoke. An important route of exposure is the circulatory system whereas blood vessels are considered to be main stream organs of Cd toxicity. Our previous results indicate that cadmium chloride (CdCl2) affects mean arterial blood pressure in hypertensive rats. We hypothesized that Cd alters the intracellular calcium transient mechanism, by cadmium-induced stimulation of MAPKs (ERK 1 & 2) which is mediated partially through calcium-dependent PKC mechanism. To investigate this hypothesis, we exposed primary cultures of vascular smooth muscle cells (VSMCs) from wistar kyoto (WKY) and spontaneously hypertensive rats (SHR) to increased concentrations of CdCl2 on cell viability, expression of mitogen-activated protein kinases (MAPKs/ERK 1 & 2), and protein kinase C (PKC) which are activated by Cd in several cell types. The results from these studies indicate that CdCl2 decreased cell viability of both SHR and WKY VSMCs in a concentration dependent-manner. Viability of both cell types decreased 33±5.3 (SHR) and 39±2.3% (WKY) when exposed to 1 μM CdCl2, whereas, 8 and 16 μM reduced viability by 66±3.1 and 62±4.5% in SHR cells. CdCl2 increased ERK 1 & 2 in a biphasic manner with maximum increase occurring when cells are exposed to 1 and 4 μM in SHR VSMCs, whereas, a reduction in ERK 1 and 2 is observed when WKY cells are treated with 2 μM. The results also indicate that CdCl2 increased PKC a/ß in both SHR and WKY VSMCs with a greater increase in expression in SHR VSMCs. In addition, the [Ca2+]i chelator, BAPTA, suppressed the CdCl2 effect, whereas, the PKC inhibitor, GF109203X, reduced the CdCl2 induced-effect on PKC expression. The present studies support the hypothesis that Cd can be a risk factor of hypertension through dysfunction of vascular smooth muscle cells under certain conditions.

Cholinergic neurons have both a low-affinity and a high-affinity choline transport process. The high-affinity choline transport is sodium dependent and thus it can be referred to as choline cotransport. Choline cotransport has been shown to be up-regulated by neuronal activity. Protein kinase C has also been shown to regulate choline cotransport. Both forms of regulation appear to modulate transport by altering the numbers of choline cotransporters in the nerve terminal membrane. The present study centers on choline cotransporter trafficking in Limulus brain hemi-slice preparations. The competitive, reversible, non-permeant ligand, [3H]hemicholinium-3, was used in binding studies to estimate the relative number of choline cotransporters in plasma membranes. The hemicholinium-3 mustard derivative has been shown to be an irreversible, highly selective, non-permeant ligand for the choline cotransporter, and was also used. Hemicholinium-3 mustard binding to the choline cotransporter blocked [3H]choline transport and [3H]hemicholinium-3 binding. Antecedent elevated potassium exposure of cholinergic tissues has been shown to up-regulate choline transport by the recruitment of additional choline cotransporters to surface membranes. This treatment was also effective in the recruitment of cotransporters following maximal inhibition by hemicholinium-3 mustard of brain hemi-slices. Long-term washout of hemicholinium-3 mustard in hemi-slices resulted in a time-dependent restoration of choline cotransport. Full recovery occurred within 2 h. In uninhibited slice preparations, both staurosporine and chelerythrine, protein kinase C inhibitors, stimulated choline uptake. However, within a 1-h washout recovery of uptake following hemicholinium-3 mustard inhibition, the staurosporine responsive but not chelerythrine responsive transport had returned. On the basis of these findings, we hypothesize the existence of two distinct populations of cycling choline cotransporters, which includes inactive or “silent” transporters.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.