Guanylin and uroguanylin: a promising nexus in intestinal electrolyte and fluid homeostasis.

Intestinal guanyl peptides like guanylin and uroguanylin are the potent regulators of fluid-ion homeostasis. They are secreted from various cells of the intestinal mucosa, including enterochromaffin cells, epithelial cells, goblet cells, Paneth cells and others. These peptide hormones serve as ligands for receptor guanylyl cyclase-C (GC-C), which produces intracellular cyclic guanosine monophosphate (cGMP) and activates protein kinase G II (PKGII). cGMP/PKGII activates cystic fibrosis transmembrane conductance regulator for anion transport to the intestinal lumen, inhibits Na+/H+ exchanger that restricts H+ secretion and Na+ absorption, resulting in the retention of luminal fluid. These functions maintain intestinal pH, prevents hypernatremia and unwanted hypervolemic shock. Additionally, fluid balance in the intestine preserves the hydrated state of the colonic mucus that influences the growth of the commensal microorganisms and bowel clearance. Moreover, GC-C/cGMP signaling is involved in the regulation of intestinal barrier integrity, epithelial cell renewal, cell cycle, DNA damage repair, inflammatory responses, epithelial-mesenchymal transition and cancer progression. Impairment of GC-C activation causes functional gastrointestinal disorders, inflammatory bowel disease, visceral pain and colorectal cancer, suggesting that oral supplementation of guanyl peptide analogs (linaclotide, plecanatide) may prove useful for the treatment of these diseases.

Leptin and obesity.

An imbalance between calorie intake and energy expenditure produces obesity. It has been a major problem in societies of the developing and developed world. In obesity an excessive amount of fat accumulates in adipose tissue cells as well as in other vital organs like liver, muscles, and pancreas. The adipocytes contain ob genes and express leptin, a 16 kDa protein. In the present communication, we reviewed the molecular basis of the etiopathophysiology of leptin in obesity. Special emphasis has been given to the use of leptin as a drug target for obesity treatment, the role of diet in the modulation of leptin secretion, and reduction of obesity at diminished level of blood leptin induced by physical exercise.

Rise of cytosolic Ca2+ and activation of membrane-bound guanylyl cyclase activity in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-01.

The cytosolic calcium level ([Ca2+]i) and the membrane-bound guanylyl cyclase activity in the isolated rat intestinal epithelial cells were investigated. Heat-stable enterotoxin of Vibrio cholerae non-01 (NAG-ST) was found to increase both the [Ca2+]i and the enzyme activity. These changes occur similarly until 5 min of incubation with NAG-ST, indicating that these changes might be involved in NAG-ST induced signal transduction in rat enterocytes.

Binding of Escherichia coli heat-stable enterotoxin and rise of cyclic GMP in COLO 205 human colonic carcinoma cells.

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Comparison of cytosolic levels of calcium and G actin in diffuse and localised adherent Escherichia coli-infected HeLa cells.

In the present study we compared the intracellular level of free calcium ([Ca2+]i) and monomeric (G)/total (G+F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC). The level of [Ca2+]i was increased in both DAEC- and LAEC-infected HeLa cells. However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca(2+)-free buffer suggested that the rise of [Ca2+]i in DAEC-infected cells was due to the influx of Ca2+ from extracellular medium, whereas Ca2+ mobilisation from the intracellular stores was responsible for the enhancement of [Ca2+]i in LAEC-infected cells. It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G/G+F actin ratio as compared to that of control cells. The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones. Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process.

Evidence for stimulation of the inositol triphosphate-Ca2+ signalling system in rat enterocytes by heat stable enterotoxin of Escherichia coli.

In Escherichia coli heat stable enterotoxin (STa)-treated rat enterocytes, the rise of inositol triphosphate (IP3) preceded the rise of [Ca2+]i. Chelation of extracellular Ca2+ with EGTA and suspension of cells in Ca2+ free buffer both demonstrated the enterotoxin-induced initial rise of [Ca2+]i with a concomitant loss of sustained phase. Furthermore, pretreatment of cells with dantrolene resulted in a decrease of the early response of [Ca2+]i, indicating the initial effect of the rise of [Ca2+]i was mostly due to its mobilization from some IP3-sensitive intracellular stores.

Alteration of the microenvironment in plasma membranes of rat enterocytes after Escherichia coli heat stable enterotoxin treatment: effect on protein kinase C activity.

Plasma membranes isolated from Escherichia coli heat stable enterotoxin (STa) treated rat enterocytes were studied in respect to protein kinase C activity and fluidity change. Pretreatment of enterocytes with STa increased the membrane bound protein kinase C activity about 5 fold as compared to control. STa treatment made the membrane more fluid as evident from a higher phospholipid/cholesterol ratio and greater unsaturated fatty acid levels. Moreover, the phase transition temperature of the STa treated membrane appeared to be significantly lower than that of the corresponding control membrane, thereby further indicating a rise in fluidity of the membrane in the former case. Our results, therefore, suggested that following STa enterotoxin treatment an appropriate fluid environment in the rat intestinal cell membrane was essential for the activation of protein kinase C.

Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli.

Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated. The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 +/- 1.69 vs 93.56 +/- 10.40 nmol/mg protein/min) and was dose dependent. Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa. The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells. Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa. However, STa had no direct role on the enzyme activity. Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes.