Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (ICCh) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited ICCh dose- dependently with IC50 of 1.1 +/- 0.1 nM (n = 6). GTPgS- induced current, however, was not inhibited by 10 nM 4-DAMP. ICCh was not recorded in pertussis- toxin (PTX)-pretreated smooth muscle cells of gastric antrum. ICCh values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 +/- 3 pA (n = 6) in the control and PTX-treated cells, respectively (P<0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that Go protein is important for activation of ACh-activated NSC channels, our results suggest that ICCh might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.

Increase of Intracellular Ca2+ Concentration Induced by Lysophosphatidylcholine in Murine Aortic Endothelial Cells

Effects of oxidized low-density lipoprotein (ox-LDL), l-alpha-stearoyl-lysophosphatidylcholine (LPC), on intracellular Ca2+ concentration were examined in mouse endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i) with fura 2-AM and reverse transcription-polymerase chain reaction (RT-PCR). LPC increased [Ca2+]i under the condition of 1.5 mM [Ca2+]o but did not show any effect under the nominally Ca2+-free condition. Even after the store depletion with 30microM 2,5-di-tert- butylhydroquinone (BHQ) or 30microM ATP, LPC could still increase the [Ca2+]i under the condition of 1.5 mM [Ca2+]o. The time required to increase [Ca2+]i (about 1 minute) was longer than that for ATP-induced [Ca2+]i increase (10-30 seconds). LPC-induced [Ca2+]i increase was completely blocked by 1microM La3+. Transient receptor potential channel(trpc) 4 mRNA was detected with RT-PCR. From these results, we suggest that LPC increased [Ca2+]i via the increase of Ca2+ influx through the Ca2+ routes which exist in the plasma membrane.