NNC-55-0396

Interstitial cells of Cajal in the plane of the myenteric plexus (ICC-MY) serve as electrical pacemakers in the stomach and small intestine. A similar population of cells is found in the colon, but these cells do not appear to generate regular slow wave potentials, as characteristic in more proximal gut regions. Ca²⁺ handling mechanisms in ICC-MY of the mouse proximal colon were studied using confocal imaging of muscles from animals expressing GCaMP6f exclusively in ICC. ICC-MY displayed stochastic, localized Ca²⁺ transients that seldom propagated between cells. Colonic ICC express ANO1 channels, so Ca²⁺ transients likely couple to activation of spontaneous transient inward currents (STICs) in these cells. The Ca²⁺ transients were due to Ca²⁺ release and blocked by cyclopiazonic acid (CPA), thapsigargin and caffeine, but unaffected by tetracaine. Antagonists of L- and T-type Ca²⁺ channels and reduction in extracellular Ca²⁺ had minimal effects on Ca²⁺ transients. We reasoned that STICs may not activate regenerative Ca²⁺ waves in ICC-MY because voltage-dependent Ca²⁺ conductances are largely inactivated at the relatively depolarized potentials of colonic muscles. We tested the effects of hyperpolarization with pinacidil, a K_ATP agonist. Ca²⁺ waves were initiated in some ICC-MY networks when muscles were hyperpolarized, and these events were blocked by a T-type Ca²⁺ channel antagonist, NNC 55-0396. Ca²⁺ waves activated by excitatory nerve stimulation were significantly enhanced by hyperpolarization. Our data suggest that colonic ICC-MY are conditional pacemaker cells that depend upon preparative hyperpolarization, produced physiologically by inputs from enteric inhibitory neurons and necessary for regenerative pacemaker activity.

Calcium channel blockers (CCBs) are commonly used as antihypertensive agents. While certain L-type CCBs exhibit antiatherogenic effects, the impact of Ca_v3.1 T-type CCBs on antiatherogenesis and lipid metabolism remains unexplored. NNC 55-0396 (NNC) is a highly selective blocker of T-type calcium channels (Ca_v3.1 channels). We investigated the effects of NNC on relevant molecules and molecular mechanisms in human THP-1 macrophages. Cholesterol efflux, an indicator of reverse cholesterol transport (RCT) efficiency, was assessed using [3H]-labeled cholesterol. In vivo, high cholesterol diet (HCD)-fed LDL receptor knockout (Ldlr^-/-) mice, an atherosclerosis-prone model, underwent histochemical staining to analyze plaque burden. Treatment of THP-1 macrophages with NNC facilitated cholesterol efflux and reduced intracellular cholesterol accumulation. Pharmacological and genetic interventions demonstrated that NNC treatment or Ca_v3.1 knockdown significantly enhanced the protein expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and liver X receptor alpha (LXRα) transcription factor. Mechanistic analysis revealed that NNC activates p38 and c-Jun N-terminal kinase (JNK) phosphorylation, leading to increased expression of ABCA1, ABCG1, and LXRα-without involving the microRNA pathway. LXRα isrequired for NNC-induced ABCA1 and ABCG1 expression. Administering NNC diminished atherosclerotic lesion area and lipid deposition in HCD-fed Ldlr^-/- mice. NNC's anti-atherosclerotic effects, achieved through enhanced cholesterol efflux and inhibition of lipid accumulation, suggest a promising therapeutic approach for hypertensive patients with atherosclerosis. This research highlights the potential of Ca_v3.1 T-type CCBs in addressing cardiovascular complications associated with hypertension.