Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi

The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models

P2 nucleotide receptors on C2C12 satellite cells

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12 family members while among P2X receptors P2X4, P2X5 and P2X7 were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca2+ ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X5 play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y2-dependent Ca2+ signaling, while the mRNA expression of P2Y2 receptor reminds still on the high level. Moreover, P2X2 receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern

Influence of Bradykinin on Diacylglycerol and Phosphatidic Acid Accumulation in Cultured Bovine Adrenal Chromaffin Cells

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [H-3]arachidonic acid or [H-3]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with P-32i for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [P-32]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [P-32]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [P-32]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C. A preliminary indication that G proteins may be involved was suggested by the observation that AlF4- stimulates [P-32]PA accumulation whereas N-ethylmaleimide inhibits bradykinin-stimulated [P-32]PA accumulation

Characterization of bradykinin-stimulated release of noradrenaline from cultured bovine adrenal chromaffin cells.

Bovine adrenal chromaffin cells were purified and maintained in culture. On exposure to bradykinin they released noradrenaline. The characteristics of this stimulus secretion were compared with the response to elevated extracellular potassium. Bradykinin released noradrenaline with an EC50 of about 2 nM, with maximum release (2-3 times control incubations) less than that elicited by high potassium or nicotine. Bradykinin analogs showed the response to have characteristics of a B2 receptor. On continuous exposure to 10 nM bradykinin the rate of release was maximal in the first 1 to 3 min, with a further slower sustained rate of release. The response is mostly, but not completely, dependent on extracellular calcium, and was inhibited by cadmium in the micromolar range. Whereas the potassium-stimulated release was highly sensitive to dihydrophyridines, the bradykinin response was not attenuated by dihydropyridine calcium channel antagonists. The results are discussed with respect to stimulus-secretion mechanisms possibly involved in the bradyininin-stimulated release in the light of previous observations characterizing a phosphoinositide response to bradykinin in these cells