Atsushi Ohsaga

Identification of Cyclic ADP-ribose-dependent Mechanisms in Pancreatic Muscarinic Ca2+ Signaling Using CD38 Knockout Mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca2+ concentration ([Ca2+] i ) using fura-2 microfluorimetry, we distinguished and characterized the Ca2+ release mechanisms responsive to cADPR. The Ca2+ response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca2+ release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca2+spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca2+ release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca2+ response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca2+signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca2+ release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca2+ spikes.

Phylogenetic, ontogenetic, and pathological aspects of the urine-concentrating mechanism.

The urine-concentrating mechanism is one of the most fundamental functions of avian and mammalian kidneys. This particular function of the kidneys developed as a system to accumulate NaCl in birds and as a system to accumulate NaCl and urea in mammals. Based on phylogenetic evidence, the mammalian urine-concentrating mechanism may have evolved as a modification of the renal medullas NaCl accumulating system that is observed in birds. This qualitative conversion of the urine-concentrating mechanism in the mammalian inner medulla of the kidneys may occur during the neonatal period. Human kidneys have several suboptimal features caused by the neonatal conversion of the urine-concentrating mechanism. The urine-concentrating mechanism is composed of various functional molecules, including water channels, solute transporters, and vasopressin receptors. Abnormalities in water channels aquaporin (AQP)1 and AQP2, as well as in the vasopressin receptor V2R, are known to cause nephrogenic diabetes insipidus. An analysis of the pathological mechanism involved in nephrogenic diabetes insipidus suggests that molecular chaperones may improve the intracellular trafficking of AQP2 and V2R, and, in the near future, such chaperones may become a new clinical tool for treating nephrogenic diabetes insipidus.

Delayed expression of large conductance K+ channels reshaping agonist‐induced currents in mouse pancreatic acinar cells

Epithelial secretory cells display cell‐specific mechanisms of fluid secretion and express large conductance voltage‐ and Ca2+‐activated K+ (Maxi‐K) channels that generate the membrane negativity for effective Cl− exit to the lumen. Rat and mouse pancreatic acinar cells had been thought to be peculiar in this sense because of the previously reported lack of Maxi‐K channels. However, this view is not entirely correct as evidenced in the present paper. Searching for their presence in pancreatic acinar cells in mice from 5 to 84 weeks of age with patch‐clamp current measurements, we demonstrated that the expression of Maxi‐K channels is regulated in an age‐associated manner after birth. The expression started at approximately 12 postnatal weeks and increased steadily up to 84 weeks. In support of this, RT‐PCR could not detect mSlo mRNA, the Maxi‐K gene, at either 7 or 8 weeks but could at 58 and 64 postnatal weeks. These results suggest that a key steering element for fluid secretion, the Maxi‐K channel, is progressively re‐organized in rodent pancreas. A pancreatic secretagogue, acetylcholine, evoked Maxi‐K channel current overlapping to various degrees on the previously known current response. This suggests that the rise in internal Ca2+ activates Maxi‐K channels which reshape the mode of secretagogue‐evoked current response and contribute to Cl− driving in fluid secretion in an age‐associated fashion.

Involvement of GTP-binding protein in pancreatic cAMP-mediated exocytosis

Abstract.We studied cAMP-mediated exocytosis in rat pancreatic acinar cells. We monitored changes in the membrane capacitance (ΔC), which reflects the granule fusion/retrieval process, with whole-cell patch-clamp capacitance measurement. The rise in cellular cAMP, caused indirectly by receptor activation by vasoactive intestinal polypeptide or directly by dibutyryl cyclic AMP, was able to induce an increase in ΔC independently of cellular Ca2+. Using the latter stimulation, we estimated the magnitude of the response to internal GTPγS [guanosine 5′-(γ-thio)trisphosphate] and/or GDPβS [guanosine 5′-(β-thio)diphosphate]. The internal GTPγS and GDPβS amplified and depressed the response, respectively. Thus, the cellular cAMP alone can trigger granule insertion independently of cellular Ca2+ and it can be controlled by cellular GTP-binding proteins, presumably those belonging to the Rab family.

Gestational Length Affects a Change in the Transepithelial Voltage and the rNKCC2 Expression Pattern in the Ascending Thin Limb of Henle's Loop

To examine whether the functional and morphologic conversion of the neonatal ascending thin limb (ATL) of Henles loop is related to gestational length, we evaluated the transepithelial voltages (Vts) of ATLs in perinatal mouse, hamster, rabbit, and rat kidneys. In isolated microperfused tubule preparations, Vts of neonatal ATLs were 23.8 ± 1.4 in mouse, 25.7 ± 2.2 in hamster, and 18.2 ± 1.6 mV in rabbit. The influence of gestational length on the Vts and rat Na-K-Cl cotransporter (rNKCC2) expression pattern was also examined in perinatal rats subjected to a prolonged gestation due to either a daily s.c. injection of 5 mg progesterone or ligation of the extremities of the uterine horn. Vts of d 3 neonates were 2.9 ± 1.0 (p < 0.0001 versus d 0); Vts of d 23 fetuses subjected to ligation were 4.9 ± 0.8 (p < 0.005 versus d 0); and Vts of d 23 fetuses given progesterone were 3.4 ± 1.7 mV (p < 0.001 versus d 0). rNKCC2 expression tended to disappear in the renal papillae of d 23 fetuses. Our data demonstrate that the perinatal conversion of the ATL is a phenomenon commonly observed among rodents; furthermore, it is dependent on the gestational length, but unrelated to the birth process.