Role of protein kinase Calpha in signaling from the histamine H(1) receptor to the nucleus

Stimulation of histamine H(1) receptors produced a marked activation of inositol phospholipid hydrolysis, intracellular calcium mobilization, and stimulation of the c-fos promoter in CHO-H1 cells expressing the H(1) receptor at a level of 3 pmol/mg protein. The latter response was determined using a luciferase-based reporter gene (pGL3). This response to histamine was not sensitive to inhibition by pertussis toxin but could be completely attenuated by the protein kinase C (PKC) inhibitor Ro-31-8220, or by 24-h pretreatment with the phorbol esters phorbol 12,13-dibutyrate or phorbol-12-myristate-13-acetate. Several isoforms of PKC can be detected in CHO-H1 cells (alpha, delta, epsilon, mu, iota, zeta) but only PKCalpha and PKCdelta were down-regulated by prolonged treatment with phorbol esters. Of the two isoforms that were down-regulated, only protein kinase Calpha was translocated to CHO-H1 cell membranes after stimulation with either histamine or phorbol esters. The PKC inhibitor G? 6976, which inhibits PKCalpha but not PKCdelta, was also able to significantly attenuate the c-fos-luciferase response to histamine. The mitogen-activated protein kinase kinase inhibitor PD 98059 markedly inhibited the response to histamine, suggesting that the likely major target for PKCalpha was the mitogen-activated protein kinase pathway. These data suggest that the histamine H(1) receptor can signal to the nucleus via PKCalpha after activation of phospholipase Cbeta.     

Desensitization of muscarinic M1 receptors of murine neuroblastoma cells (clone N1E-115) without receptor down-regulation and protein kinase C activity

Acute desensitization of M1 muscarinic receptor-mediated responses (cyclic GMP formation and inositol phosphate release) was studied in murine neuroblastoma cells (N1E-115 clone). After a 45-min incubation at 37 degrees of N1E-115 cells either in monolayer or in suspension, with the muscarinic agonist carbachol (1 mM), the receptor-mediated cyclic GMP response to carbachol was nearly completely lost. This loss was associated with greater than 80% loss of carbachol-mediated inositol phosphate release. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) inhibited both responses with similar potencies. Carbachol or PMA reduced by 30-40% the number of muscarinic receptor sites for antagonist and agonist on intact cells (determined in binding assays using [3H]N-methylscopolamine) only for cells in monolayer and not for those in suspension. PMA but not carbachol pretreatment of cells in monolayer or in suspension caused a translocation of [3H]phorbol 12,13-dibutyrate binding and protein kinase C activity. In addition, desensitization to carbachol occurred in cells largely depleted of protein kinase C by chronic exposure to PMA. Thus, agonist-mediated down-regulation is not needed for muscarinic M1 receptor desensitization, which may be a result of the activation of a receptor-activated kinase different from protein kinase C.     

Desensitization of histamine H1 receptor-mediated cyclic GMP production in guinea-pig lung.

Histamine H1 receptor-mediated production of cGMP in guinea-pig lung tissue becomes rapidly desensitized after previous exposure to histamine. This desensitization is clearly concentration dependent and appears to be homologous. Responses to histamine are also inhibited by previous treatment with phorbol 12,13-dibutyrate. Yet, the time course of the inhibition is considerably slower and the maximal inhibition is significantly less compared to receptor desensitization. Moreover, the effects of the phorbol ester are not confined to H1 receptor responses. Since the effects of receptor desensitization are also not prevented by several protein kinase C inhibitors, the development of homologous H1 receptor desensitization is not dependent upon protein kinase C activation, but is caused by a yet unidentified mechanism.     

Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells

1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.     

Homologous and heterologous phosphorylations of human histamine H1 receptor in intact cells

Homologous and heterologous phosphorylations of histamine H1 receptor (H1R) in intact cells were investigated using Chinese hamster ovary cells stably co-expressing c-myc-tagged human histamine H1 and muscarinic M3 receptors. Increase in histamine-induced homologous phosphorylation of H1R was induced in a dose- and time-dependent manner. Maximum phosphorylation of H1R by 8-fold over the basal level was induced 1 min after the stimulation, and the increased phosphorylation level was maintained over 40 min. M3 receptor-mediated heterologous phosphorylation of H1R reached maximum by 2-fold over the basal level at 5 min after the stimulation and then rapidly returned to the basal level by 40 min after the stimulation. Histamine-induced phosphorylation of H1R was partially inhibited by three protein kinase inhibitors including Ro-31-8220 for protein kinase C (PKC), KN-93 for calcium/calmodulin-dependent kinase II (CaMKII), and KT5823 for protein kinase G (PKG), while, M3-receptor-mediated phosphorylation of H1R was completely inhibited by Ro 31-8220. Protein kinase activators including phorbol 12-myristate 13-acetate (PMA), 8-bromo-cyclic GMP (8-Br-cGMP), and 8-bromo-cyclic AMP (8-Br-cAMP) induced increases in H1R phosphorylation. Increased phosphorylation of H1R, by 5-fold over the basal level, induced with a combination of PMA, 8-Br-cGMP, and 8-Br-cAMP was still lower than that with histamine. It was suggested that H1R-mediated H1R phosphorylation involves the activation of PKC, CaMKII, PKG, and other unidentified kinases including G-protein coupled receptor kinases (GRKs) and that PKC is solely involved in M3 receptor-mediated H1R phosphorylation.     

Desentization of histamine H1 receptor-mediated cyclic GMP production in guinea-pig lung

Histamine H₁ receptor-mediated production of cGMP in guinea-pig lung tissue becomes rapidly desensitized after previous exposure to histamine. This desensitization is clearly concentration dependent and appears to be homologous. Responses to histamine are also inhibited by previous treatment with phorbol 12,13-dibutyrate. Yet, the time course of the inhibition is considerably slower and the maximal inhibition is significantly less compared to receptor desensitization. Moreover, the effects of the phorbol ester are not confined to H₁ receptor responses. Since the effects of receptor desensitization are also not prevented by several protein kinase C inhibitors, the development of homologous H₁ receptor desensitization is not dependent upon protein kinase C activation, but is caused by a yet unidentified mechanism.     

Identification of protein kinase C phosphorylation sites involved in phorbol ester-induced desensitization of the histamine H1 receptor

The histamine H1 receptor (H1R)-mediated signaling cascade is inhibited by phorbol ester-induced protein kinase C (PKC) activation. Cloning studies of the H1Rs have shown that several potential PKC phosphorylation sites are located in the third intracellular loop of H1R. To elucidate the molecular mechanism of PKC-mediated desensitization, we identified amino acid residues that are involved in the desensitization of the H1R. Two amino acid residues (Ser396, Ser398) were determined to be PKC phosphorylation sites by in vitro phosphorylation studies using a series of synthetic peptides. Treatment with phorbol ester decreased histamine-induced accumulation of inositol phosphates in Chinese hamster ovary cells expressing the H1R with a rightward shift in the EC50 value, which implies the uncoupling of the receptor from the G protein. Site-directed mutagenesis studies showed that substitution of alanine for Ser398 but not for Ser396 markedly attenuated the effect of phorbol ester, which suggests that the Ser398 residue was primarily involved in PKC-mediated desensitization.     

Protein kinase C activators sensitize cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Pretreatment of 1321N1 human astrocytoma cells with phorbol 12-myristate-13-acetate or other activators of protein kinase C led to 2.5- to 5-fold increases (sensitization) in subsequent stimulation by forskolin of intracellular cyclic AMP accumulation. These compounds caused much smaller or no increases in receptor-mediated stimulation of cyclic AMP accumulation induced by isoproterenol and by prostaglandin E1. Carbachol and histamine, agonists acting at receptors coupled to polyphosphoinositide turnover in these cells, induced less sensitization of subsequent stimulation by forskolin but greater sensitization of stimulation by isoproterenol and by prostaglandin E1. The specificities of various analogs of phorbol 12-myristate-13-acetate, for induction of sensitization of forskolin stimulation were consistent with involvement of protein kinase C. The effects of protein kinase inhibitors and of down-regulation of protein kinase C activity also indicated involvement of protein kinase C in sensitization of forskolin stimulation, although additional mechanisms are likely to be involved in sensitization of isoproterenol stimulation. Neither pertussis toxin pretreatment nor inclusion of isobutylmethylxanthine during assays of cyclic AMP accumulation were able to prevent or mimic these sensitization phenomena, suggesting that the primary site of modification responsible for sensitization is neither the inhibitory guanine nucleotide-binding protein nor cyclic AMP phosphodiesterase. Sensitization was only observed in assays with intact cells. These results, together with those from our previous study describing protein kinase C-mediated desensitization of broken cell adenylate cyclase activity, indicate that activation of protein kinase C leads to multiple changes in the receptor-stimulated adenylate cyclase signal transduction pathway of these cells.     

Desensitization by histamine of H2 receptor activity in HGT-1 human cancerous gastric cells

Histamine produced a time-dependent (half-life: 20 min at 37 degrees C), temperature-dependent (no effect at 20 degrees C) and homologous desensitization of histamine H2 receptor activity (H2 R) in HGT-1 cells. Maximal and half-maximal desensitization were respectively observed at 10(-5) and 2 X 10(-7) M histamine. Decline of responsiveness in intact cells was related to a remarkable loss in histamine efficacy (from 15- to 2-fold stimulation in control and treated cells). The affinity of the H2R for histamine (EC50 = 10(-5) M) did not change during desensitization. Paradoxically, histamine treatment is associated with increased [3H] histamine binding capacity in intact HGT-1 cells, and no change in H2 receptor antagonist binding ([3H]-tiodine and [3H]-SKF 93479). Desensitization process was preferentially mimicked by H2 receptor agonists (impromidine greater than histamine greater than AET greater than PEA) and preferentially reversed by simultaneous addition of H2 receptor antagonists (cimetidine greater than DPH). We suggest that the desensitization of H2R activity by histamine presented here may be involved in the pathophysiological regulation and pharmacological control of gastric cell function in man.     

Inhibition of histamine H1 receptor downregulation by KT5823, a protein kinase G inhibitor

The role of various protein kinases in the downregulation of histamine H(1) receptors was studied by using their inhibitors and activators. Human histamine H(1) receptors (H(1)Rs) expressed in CHO cells were downregulated by histamine in a dose- and time-dependent manner, and this downregulation continued to increase over a 24-h period. KT5823, an inhibitor of protein kinase G, remarkably but not completely reversed the histamine-induced H(1)R downregulation over 24 h. HA1004, another inhibitor of protein kinase G, showed a similar inhibitory effect. However, both 8-Br-cGMP and 8-pCPT-cGMP, membrane-permeable analogues of cGMP, did not show any effects on H(1)R downregulation in the absence or presence of histamine. Ro 31-8220, an inhibitor of protein kinase C (PKC), did not affect histamine-induced downregulation of H(1)R; nor did phorbol 12-myristate 13-acetate, a PKC-activating phorbol ester. Similarly, histamine-induced downregulation of H(1)R was unaffected by either H-89, an inhibitor of protein kinase A, or 8-Br-cAMP, a membrane-permeable analogue of cAMP.     

Agonists and phorbol esters desensitize beta-adrenergic receptors by different mechanisms

Exposure of 1321N1 human astrocytoma cells to the protein kinase C (PKC) activator phorbol 12-myristate, 13-acetate (PMA) led to a rapid and concentration-dependent decrease in isoproterenol (ISO)-stimulated adenylate cyclase (AC) activity in cell lysates. This desensitization of beta-adrenergic receptor (BAR) function was mimicked by mezerein, which also activates PKC, but not by 4-O-methyl-PMA, which is a very weak activator of PKC. Pretreatment with PMA led to desensitization of AC activity stimulated by ISO and by prostaglandin E1, in contrast to the beta-receptor-specific desensitization induced by ISO. Stimulation of AC activity by forskolin and by fluoride remained unaltered. The extent of desensitization observed with PMA plus ISO was greater than with either agent alone. Desensitization with PMA did not result in internalization of BAR, as assessed by sucrose density gradient centrifugation assays and by assays of competition by the hydrophilic ligand ISO for radioligand binding to intact cell receptors. PMA pretreatment did not alter the apparent affinity of the agonist ISO for intact cell BAR, nor was the potency of ISO for stimulation of AC activity altered. The protein kinase inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited the desensitization induced by PMA but not that induced by ISO. These results indicate that activation of PKC can lead to desensitization of receptor-stimulated AC activity but that agonist-induced desensitization of BAR-stimulated AC activity occurs by a different mechanism.     

Role of H1 receptors in histamine-mediated up-regulation of STAT4 phosphorylation

Histamine shifts TH1/TH2 cytokine balance from TH1 to TH2 cytokines and regulates the function of lymphocytes after binding to histamine receptors. The phosphorylation of STAT factors and the translocation to the nucleus are important steps in the regulation of TH1/TH2 cytokine balance. This study was designed to investigate the effects of histamine on the phosphorylation of STAT4. C57BL/6 splenocytes were isolated and treated with histamine (10(-4) to 10(-9) M) after activation with either PMA (phorbol 12 myristate 13-acetate) plus ionomycin or IL-12. The phosphorylated STAT4 levels were analyzed by Western Blot Analysis. Unstimulated splenocytes expressed both STAT4 and phosphorylated STAT4. However, phosphorylated STAT4 gradually declined within 24 h. Histamine increased the phosphorylation of STAT4 at lower concentrations (10(-6) to 10(-9) M), and had no effect at higher concentrations (10(-4) and 10(-5) M) after the cells were stimulated with PMA + ionomycin. Histamine did not affect IL-12-induced phosphorylation of STAT4. To characterize the histamine receptor subtypes involved in the up-regulation of STAT4 phosphorylation, various H1, H2 and H3/H4 receptor antagonists and/or agonists were employed. H1 receptor agonist (betahistine), but not H2 receptor agonist (amthamine), induced phosphorylation of STAT4. H1 receptor antagonist (pyrilamine) inhibited histamine-mediated phosphorylation of STAT4. However, H2 receptor antagonist (ranitidine) and H3/H4 receptor antagonist (thioperamide) did not alter this effect. Tyrosine kinase inhibitor (tyrphostin) failed to block histamine-mediated phosphorylation of STAT4. These observations suggest that histamine up-regulated the phosphorylation of STAT4 via H1 receptors, and that the Ca2+-PKC pathway, but not the tyrosine kinase pathway, was involved in this effect.     

Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C.

1. The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1- and ATP-receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF-2). Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1- or ATP-receptor activation. 2. Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester beta-phorbol-12,13 dibutyrate (PDBu; 1 microM) attenuated histamine (100 microM) and ATP (10 microM)-induced release of intracellular Ca2+ and Ca2+ influx. 3. The selective PKC inhibitor, Ro 31-8220 (10 microM), reversed the PDBu-induced attenuation of histamine (100 microM)-stimulated Ca2+ responses. 4. Histamine H1- and ATP-receptors are readily susceptible to homologous desensitization since short-term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1-receptor activation-induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa. 5. Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31-8220 (10 microM). 6. These data suggest that PKC activation can attenuate histamine H1- and ATP-receptor mediated Ca2+ responses. However, based on our experimental data, PKC-independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1- and ATP-receptor mediated Ca2+ responses in DDT1MF-2 cells.     

Activators of protein kinase C and cyclic AMP-dependent protein kinase regulate intracellular calcium levels through distinct mechanisms in mouse anterior pituitary tumor cells

The effects of the protein kinase C activator, phorbol myristate acetate (PMA), on cytosolic calcium levels and adrenocorticotropin (ACTH) release from the mouse anterior pituitary tumor cell line, AtT-20, were compared to those induced by the hormone, corticotropin-releasing factor (CRF), a stimulant of cAMP-dependent protein kinase activity. Cytosolic calcium levels were measured using the fluorescence probe Quin 2. PMA induced a time- and concentration-dependent rise in cytosolic calcium levels and ACTH release from AtT-20 cells that was blocked by verapamil and nifedipine, antagonists of voltage-regulated calcium channels, and tetraethylammonium (TEA), a K+ channel antagonist. The inactive phorbol ester, 4-phorbol 12,13-didecanoate, did not alter cytosolic calcium levels or ACTH release. Several minutes after the initial stimulation of calcium influx by PMA, cytosolic calcium levels returned to basal levels despite the continued presence of the phorbol ester. A short pretreatment (2-4 min) of AtT-20 cells with PMA abolished the ability of K+, CRF, and forskolin to raise intracellular calcium levels. These findings indicate that phorbol esters induce a secondary inhibition of calcium influx after an initial stimulation. In contrast to the effects of PMA, CRF induced a sustained rise in cytosolic calcium levels and did not reduce the subsequent stimulation of calcium influx by K+ or PMA. CRF-stimulated calcium influx was blocked by verapamil but not TEA. The ability of CRF to elevate cytosolic calcium levels was mediated by cAMP-dependent protein kinase because the insertion of a synthetic peptide inhibitor of cAMP-dependent protein kinase activity into AtT-20 cells attenuated the ability of CRF and forskolin but not PMA to raise cytosolic calcium levels. The results suggest that activators of protein kinase C and cAMP-dependent protein kinase regulate intracellular calcium levels in AtT-20 cells through different mechanisms.     

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a.

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.     

Identification of protein kinase C phosphorylation sites involved in desensitization of the histamine H1 receptor

We studied whether direct phosphorylation plays a key role in protein kinase C-activating phorbol ester-mediated H1 receptor desensitization. Several potential protein kinase C-mediated phosphorylation sites were located in the third cytoplasmic loop form our cloning studies of H1 receptors. Ser396 and Ser398 were determined to be the phosphorylation sites by in vitro phosphorylation studies using synthetic peptides corresponding to the partial amino acid sequence of the third cytoplasmic loop. Mutant H1 receptors whose Ser396 or Ser398 were displaced by alanine were expressed in Chinese hamster ovary cells by site-directed mutagenesis. Characterization of these receptors revealed that Ser398, but not Ser396, was primarily responsible for protein kinase C-mediated H1 receptor desensitization.     

Regulation of the human histamine H1 receptor stably expressed in Chinese hamster ovary cells.

1. The human H1 receptor gene expressed in Chinese hamster ovary cells (CHOhumH1) encodes a classical histamine H1 receptor with a pharmacology similar to that of the H1 receptor found in guinea-pig cerebellum and the endogenously expressed human H1 receptor in 1321N1 astrocytoma cells as determined by [3H]-mepyramine binding studies. 2. In CHOhumH1 cells, histamine induced a concentration-dependent rise in inositol phosphates (EC50 2.23 +/- 0.97 microM) and a rapid increase of [Ca2+]i, followed by a sustained increase of [Ca2+]i upon addition of 100 microM histamine. 3. Short-term exposure of CHOhumH1 cells to histamine (100 microM) resulted in a decrease of subsequent histamine-induced Ca2+ responses. The histamine-induced desensitization appeared to be heterologous as the ATP-induced Ca2+ response was also found to be affected. 4. The process of heterologous histamine-induced desensitization of the Ca2+ response in CHOhumH1 cells can be ascribed to an alteration at the level of the intracellular Ca2+ pool, as the Ca2+ response of caffeine (10 mM), which releases Ca2+ from intracellular Ca2+ stores was also attenuated upon short-term histamine exposure. 5. In CHOhumH1 cells the PKC activator, PMA, was found to inhibit the histamine (100 microM)-induced Ca2+ response concentration-dependently (IC50 0.2 +/- 0.03 microM) as well as the ATP (100 microM)-induced Ca2+ response. However, this inhibition was only partial and less effective than histamine-pretreatment. Moreover, in CHOhumH1 cells PKC downregulation induced by long-term exposure to PMA (1 microM) did not affect the histamine-induced desensitization nor did pretreatment with the specific PKC inhibitor Ro-31-8220 (10 microM), indicating that in CHOhumH1 cells PKC is probably not involved in the heterologous desensitization. 6. Long-term treatment of CHOhumH1 cells with histamine or other H1 agonists resulted in a time- and concentration-dependent decrease in the number of H1 receptor binding sites (maximal reduction: 47 +/- 5%). 7. Long-term exposure of CHOhumH1 cells to ATP or PMA did not affect H1 receptor density. 8. Both histamine (100 microM)- and ATP (100 microM)-induced Ca2+ responses were affected upon long-term exposure of cells to histamine (100 microM), which might be explained by an alteration at a level distant from the receptor. 9. These results show that in CHOhumH1 cells the human histamine H1 receptor is susceptible to short-term and long-term receptor regulation in which PKC does not seem to play a role. The CHOhumH1 cells therefore provide an excellent model system for studying the mechanism(s) of PKC-independent H1 receptor regulation.     

Clathrin-independent internalization of the human histamine H1-receptor in CHO-K1 cells

The aim of the present study was to investigate the cellular pathway involved in histamine-stimulated internalization of the human H1-receptor in CHO-K1 cells expressing N-terminal myc-tagged H1-receptor (Myc-H1) or N-terminal myc-C-terminal green fluorescent protein (Myc-GFP H1) versions of the receptor. Studies of 3H-mepyramine binding and histamine-stimulated 3H-inositol phosphate accumulation in these cells showed that the Myc-H1 and Myc-GFP H1-receptors had identical pharmacology to the wild-type H1-receptor. The Myc-H1-receptor was rapidly internalized in CHO-K1 cells following stimulation with histamine (0.1 mM). This response occurred within 15 min, and could be prevented by the quaternary H1-receptor antagonist alpha-pirdonium. Similar data were obtained with the Myc-GFP H1-receptors. Internalization of the Myc-GFP H1-receptor was maintained in the absence of extracellular calcium and was not inhibited by the CAM kinase II inhibitor KN-62 (10 microM). Phorbol dibutyrate, an activator of protein kinase C, was also able to stimulate internalization of the H1-receptor. However, inhibition or downregulation of protein kinase C (which significantly modified histamine-stimulated inositol phosphate responses) was without effect on the internalization of the H1-receptor stimulated by histamine. Hypertonic sucrose did not prevent histamine-induced internalization of the Myc-GFP H1-receptor, but was able to attenuate internalization of transferrin via clathrin-mediated endocytosis in the same cells. In contrast, preincubation of cells with filipin or nystatin, which disrupts caveolae and lipid rafts, completely inhibited the histamine-induced internalization of the Myc-GFP H1-receptor, but was without effect on the sequestration of transferrin. The H1-receptor and cholera toxin subunit B were colocalized under resting conditions at the cell surface. Immunohistochemical studies with an antibody to caveolin-1 confirmed that this protein was also localized predominantly to the plasma membrane. However, following stimulation of CHO-Myc-GFP H1 cells with histamine, there was no evidence for internalization of caveolin-1 in parallel with the H1-receptor.These data provide strong evidence that the H1-receptor is internalized via a clathrin-independent mechanism and most likely involves lipid rafts.