Satyanarayan Bhat

Sensitization and translocation of TRPV1 by insulin and IGF-I

Insulin and insulin-like growth factors (IGFs) maintain vital neuronal functions. Absolute or functional deficiencies of insulin or IGF-I may contribute to neuronal and vascular complications associated with diabetes. Vanilloid receptor 1 (also called TRPV1) is an ion channel that mediates inflammatory thermal nociception and is present on sensory neurons. Here we demonstrate that both insulin and IGF-I enhance TRPV1-mediated membrane currents in heterologous expression systems and cultured dorsal root ganglion neurons. Enhancement of membrane current results from both increased sensitivity of the receptor and translocation of TRPV1 from cytosol to plasma membrane. Receptor tyrosine kinases trigger a signaling cascade leading to activation of phosphatidylinositol 3-kinase (PI(3)K) and protein kinase C (PKC)-mediated phosphorylation of TRPV1, which is found to be essential for the potentiation. These findings establish a link between the insulin family of trophic factors and vanilloid receptors.

Osmotic diuretics induce adenosine A1 receptor expression and protect renal proximal tubular epithelial cells against cisplatin-mediated apoptosis.

Osmotic diuretics are used successfully to alleviate acute tubular necrosis (ATN) produced by chemotherapeutic agents and aminoglycoside antibiotics. The beneficial action of these agents likely involves rapid elimination of the nephrotoxic agents from the kidney by promoting diuresis. Adenosine A1 receptor (A1AR) subtype present on renal proximal tubular epithelial and cortical collecting duct cells mediates the antidiuretic and cytoprotective actions of adenosine. These receptors are induced by activation of nuclear factor (NF)-κB, a transcription factor reported to mediate hyperosmotic stress-induced cytoprotection in renal medullary cells. In this study, we tested the hypothesis that induction of the A1AR in renal proximal tubular cells by NF-κB contributes to the cytoprotection afforded by osmotic diuretics. Exposure of porcine renal proximal tubular epithelial (LLC-PK1) cells to mannitol or NaCl produced a significant increase in A1AR. This increase was preceded by adenosine release and NF-κB activation. Expression of an IκB-α mutant, which acts as a superrepressor of NF-κB, abrogated the increase in A1AR. Cells exposed to mannitol demonstrated increased reactive oxygen species (ROS) generation, which was attenuated by inhibiting xanthine oxidase with allopurinol. Allopurinol attenuated both the increase in A1AR expression and NF-κB activation produced by osmotic diuretics, indicating a role of adenosine metabolites in these processes. Treatment of LLC-PK1 cells with cisplatin (8 μm) resulted in apoptosis, which was attenuated by mannitol but exacerbated by selective A1AR blockade. Administration of mannitol to mice increases A1AR expression and activation of NF-κB in renal cortical sections. Taken together, these data provide novel mechanisms of nephroprotection by osmotic diuretics, involving both activation and induction of the A1AR, the latter mediated through activation of a xanthine oxidase pathway leading to ROS generation and promoting activation of NF-κB.

Cisplatin up-regulates the adenosine A1 receptor in the rat kidney

Cisplatin, a widely used anticancer drug, produces significant oto- and nephrotoxicity. Previous data from our laboratory, using cultured cell lines, indicated that cisplatin increases the expression of the adenosine A(1) receptor subtype through generation of reactive oxygen species and activation of nuclear factor-kappa B (NF-kappa B). Since the adenosine A(1) receptor plays an important role in normal renal physiology, this study was performed to determine whether cisplatin modulates adenosine A(1) receptor expression in vivo and whether these receptors play a role in the nephrotoxicity. Male Sprague-Dawley rats, treated with cisplatin (8 mg/kg), developed nephrotoxicity within 3 days, as demonstrated by increased serum creatinine and blood urea nitrogen. Cisplatin also produced a significant increase in malondialdehyde, apoptosis and necrosis in the kidney. The above changes were associated with a time-dependent increase in the expression of adenosine A(1) receptor, as determined by radioligand binding assays, Western blotting and immunocytochemistry, and an increase in adenosine A(1) receptor transcripts. Administration of selective and nonselective antagonists of the adenosine A(1) receptor produced either no change or exacerbated the nephrotoxicity produced by cisplatin. These data indicate that cisplatin can regulate the adenosine A(1) receptor in the kidney and suggest a cytoprotective role of this receptor subtype against cisplatin-induced nephrotoxicity.

Cisplatin up-regulates adenosine A1 receptors in rat testes

Reactive oxygen species contribute to male infertility by reducing sperm function. Our laboratory has recently demonstrated that reactive oxygen species stimulate the expression of adenosine A(1) receptor which confers cytoprotection in a variety of tissues. Since the adenosine A(1) receptor is highly expressed in the testis, the goal of this study was to determine whether this testicular adenosine A(1) receptor could also be regulated in vivo by reactive oxygen species. Cisplatin, a chemotherapeutic agent shown to alter testicular function, was used to generate reactive oxygen species in vivo. Testes obtained from Sprague-Dawley rats treated with cisplatin (8 mg kg(-1)) demonstrate increased lipid peroxidation and induction of heat shock protein by day 3. In addition, radioligand binding and Western blotting studies indicate an increase in testicular adenosine A(1) receptor in these rats. Scatchard analysis of [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) binding data indicates a significant increase in adenosine A(1) receptor number (B(max)) from 309+/-77 to 540+/-69 fmol mg(-1) protein in the cisplatin-treated group. The respective equilibrium dissociation constants (K(d)s) were 3.2+/-1.5 and 3.0+/-0.7 nM for the control and cisplatin-treated groups, respectively. Northern blotting analysis of rat testicular poly (A)(+) RNA indicates two adenosine A(1) receptor transcripts migrating at 3.4 and 5.6 kb, whose combined levels were increased by 49.3+/-9.3% following cisplatin treatment. These results indicate that cisplatin enhances adenosine A(1) receptor expression in the rat testis, possibly through promotion of oxidative stress.

Inducibility of HBD-2 in acute burns and chronic conditions of the lung

The respiratory tract produces a number of molecules that act in the first line of host defense to protect against pathogenic colonization and tissue invasion. Most of the innate antimicrobial activity can be attributed to airway fluid proteins, such as lysozyme, lactoferrin, and secretory leukoproteinase inhibitor, and peptides, such as defensins. Human beta-defensins are cationic antimicrobial peptides with broad and potent microbicidal activity that have been shown to play a role in protecting the healthy lung from infection. To determine the effect of thermal injury on the production of the inducible beta-defensin, human beta-defensin-2 (HBD-2), we measured the concentration of HBD-2 by Western blot analysis in bronchoalveolar lavage samples from the lungs of burned patients with and without inhalation injury. Our data demonstrates an increased amount of HBD-2 in the pulmonary airways with thermal injury compared to normal lung. A further substantial increase in levels was noted in chronic lung conditions.