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Chimeric G proteins made by replacing the COOH-terminal heptapeptide of [G.sub.[alpha]]q with the COOH-terminal heptapeptide of [G.sub.[alpha]]s or [G.sub.[alpha]]i were used to assess the relative coupling of [[beta].sub.3]-adrenergic receptor ([[beta].sub.3]-AR) splice variants ([[beta].sub.3A] and [[beta].sub.3B]) to [G.sub.[alpha]]s and [G.sub.[alpha]]i. The [G.sub.[alpha]]q/s and [G.sub.[alpha]]q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium ([Ca.sup.2+]i). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat [[beta].sub.3]-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, [[beta].sub.3]-AR-induced coupling to [G.sub.[alpha]]q/s produced a rapid eightfold increase in [Ca.sup.2+]i followed by a slow decay to levels 25% above baseline. [G.sub.[alpha]]q/i also linked rat [[beta].sub.3]-AR to mobilization of [Ca.sup.2+]i in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in [Ca.sup.2+]i was only 30% of the response obtained with [G.sub.[alpha]]q/s. Activation of the rat [[beta].sub.3]-AR also increased GTP binding to endogenous [G.sub.[alpha]]i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse [[beta].sub.3A]- and [[beta].sub.3B]-AR to [G.sub.[alpha]]i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to [G.sub.[alpha]]q/s and to increases in intracellular cAMP through endogenous [G.sub.[alpha]]s. The [[beta].sub.3A]- and [[beta].sub.3B]-AR coupled equivalently to [G.sub.[alpha]]q/i, but the temporal patterns of [Ca.sup.2+]i mobilization indicated that coupling was significantly less efficient than coupling to [G.sub.[alpha]]q/s. Collectively, these findings indicate less efficient but equivalent coupling of [[beta].sub.3A]- and [[beta].sub.3B]-AR to [G.sub.[alpha]]i vs. [G.sub.[alpha]]s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to [[beta].sub.3]-AR activation. [beta]-adrenergic receptor; G proteins; cyclic adenosine monophosphate; signaling plasticity

Siberian hamsters (Phodopus sungorus) exhibit a naturally occurring, reversible seasonal obesity with body fat peaking in long 'summerlike' days (LDs) and reaching a nadir in short 'winterlike' days (SDs). These SD-induced decreases in adiposity are mediated largely via sympathetic nervous system (SNS) innervation of white adipose tissue (WAT), as indicated by increased WAT norepinephrine (NE) turnover. We examined whether SDs also increase sensitivity to NE-stimulated lipolysis. This was accomplished by measuring NE- and [[beta].sub.3]-adrenoceptor ([[beta].sub.3]-AR) agonist (BRL-37344)-induced lipolysis (glycerol release) as well as NE-induced cAMP accumulation by inguinal, epididymal, and retroperitoneal WAT (IWAT, EWAT, and RWAT) in isolated adipocytes of LD- and SD-housed hamsters. SDs increased potency/efficacy of NE-triggered lipolysis in a temporally and fat pad-specific manner. Thus when WAT pad mass decreased most rapidly (5 wk of SDs), potency (sensitivity/E[C.sub.50]) and efficacy (maximal response asymptote) of NE-stimulated lipolysis were increased for all WAT pads and also at 10 wk for IWAT compared with their LD counterparts. SD enhancement of lipolysis was similar for NE and BRL-37344 in IWAT adipocytes. These results, coupled with our previous demonstration that SDs upregulate WAT [[beta].sub.3]-AR mRNA expression, suggest that increased [[beta].sub.3]-ARs mediated the SD-induced increased NE sensitivity. NE-stimulated adipocyte accumulation of cAMP was greater after 5 wk of SDs for IWAT and EWAT and after 10 wk of SDs for IWAT compared with LDs, with no photoperiod effect for RWAT. Therefore, the SD-induced increase in SNS drive to WAT and increased sensitivity to this drive may work together to increase lipolysis in SDs. lipolysis; body fat; BRL-37344; body weight; sympathetic

The role of electroneutral Na+/H+ exchange (NHE) in extracellular acidification and hormone secretion was investigated. Results indicated that stimulation of cholecystokinin (CCK) secretion is functionally linked with activation of NHE through a mechanism that may involve pH-sensitive changes in membrane potential. It was proposed that inhibition of NHE lowers intracellular pH and causes membrane depolarization through the effects of H+ on K+ permeability.

Nitric oxide acts as an important regulator of cholecystokinin (CCK) release in the gastrointestinal tract. NO activates the potassium channels that promote CCK secretion by increasing the levels of cyclic guanosine monophosphate in STC-1 cell lines. A parallel pathway for NO-mediated stimulation of adenosine triphosphate-sensitive K channels was suggested due to the inability of NO-generating agents to activate the channel. Furthermore, low levels of NO led to the depolarization of K channel basal membrane potential.

The role of beta-adrenergic receptors (beta-AR) in the regulation of cholescystokinin (CCK) secretion in the CCK secreting cell line, STC-1, was investigated. Photolabeling studies and addition of isoproteronol into the solution showed increases in cAMP levels and CCK release, indicating a possible association between the two compounds. Cells treated with L-type calcium blocker diltiazem inhibited CCK secretion. These results suggest that beta-ARs are present in STC-1 cells and associated with cAMP production.

Experimental observations including patch-clamp studies suggest a calcium-dependent pathway for the secretion of cholecystokinin (CCK) in STC-1 cells, stimulated by phenylalanine. Ten micromoles of diltiazem blocks this phenylalanine-induced CCK secretion. Pertussis toxin does not regulate this dose-dependent CCK release.

A study of the action of calcium in the bombesin-activated secretion of cholecystokinin (CCK) from intestinal CCK-generating cell-line (STC-1) through radioimmunoassay reveals that the secretion occurs through binding to a receptor and is influenced by enhanced (Ca2+)i. STC-1 cell line may facilitate the analysis of the modulation of intestinal CCK release. Bombesin induces a dose-dependent secretion of CCK, which requires calcium intake.

Experimental studies of the role of potassium channel activity in the secretion of cholecystokinin (CCK) in STC-1 cells reveal that the activity of basally active ATP-sensitive potassium channels tonically controls CCK release. Addition of glucose inhibits the channel activity, leading to depolarization of the plasma membrane and an increase in the cystolic calcium. The glucose-induced calcium release induces the secretion of CCK and a calcium channel blocker inhibits the glucose-induced CCK release. Patch-clamp and 86Rb efflux studies reveal the endogenous expression of ATP-sensitive potassium channels in STC-1 cells.

Capsaicin vanilloid receptor-1 mediates substance P release in experimental pancreatitis. Am J Physiol Gastrointest Liver Physiol 281: G1322-G1328, 2001.--We examined whether the capsaicin vanilloid receptor-1 (VR1) mediates substance P (SP) release from primary sensory neurons in experimental pancreatitis. Pancreatitis was achieved by 12 hourly injections of caerulein (50 [micro]g/kg ip) in mice. One group received capsazepine (100 [micro]mol/kg sc), a competitive VR1 antagonist, at 4-h intervals. Neurokinin-1 receptor (NK1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK1R endocytosis. The severity of pancreatitis was assessed by measurements of serum amylase, pancreatic myeloperoxidase (MPO) activity, and histological grading. Caerulein administration caused significant elevations in serum amylase and pancreatic MPO activity, produced histological evidence of pancreatitis, and caused a dramatic increase in NK1R endocytosis. Capsazepine treatment significantly reduced the level of NK1R endocytosis, and this was associated with similar reductions in pancreatic MPO activity and histological severity of pancreatitis. These results demonstrate that repeated caerulein stimulation causes experimental pancreatitis that is mediated in part by stimulation of VR1 on primary sensory neurons, resulting in endogenous SP release. capsazepine; neurogenic inflammation; neurokinin-1 receptor; pancreatic acinar cells; primary sensory neurons