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R H Kramer

Publications and source records attributed to R H Kramer.

At least 73 records · Page 4Linked to original sources
PubMed1992

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.

Amino Acid Sequence↗
PubMed1992

Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons.

In olfactory receptor neurons, odorants stimulate production of cAMP, which directly activates cyclic nucleotide-gated (CNG) channels. Olfactory adaptation is thought to result from a rise in intracellular Ca2+. To determine whether inhibition of CNG channels plays a role in adaptation, we have investigated the action of Ca2+ on these channels in inside-out "macro" patches from the dendrite and cilia of catfish olfactory neurons. Internal Ca2+, with a K1/2 of 3 microM, profoundly inhibits CNG channels by shifting the dose-response relationship to higher cAMP levels without altering the maximal response. The inhibition does not appear to result from a direct interaction of Ca2+ with the CNG channel. Thus, the inhibition washes out after excision of the inside-out patch, and Ca2+ does not inhibit the cloned catfish CNG channel expressed in Xenopus oocytes. Hence we propose that a regulatory Ca(2+)-binding protein, distinct from the CNG channel, controls the gain of signal transduction and contributes to olfactory adaptation by decreasing the sensitivity of the CNG channel to cAMP.

Animals↗
PubMed1992

Integrin receptors and RGD sequences in human keratinocyte migration: unique anti-migratory function of alpha 3 beta 1 epiligrin receptor.

The migration of keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. However, the mechanisms by which keratinocytes migrate over extracellular matrix components are unknown. In this study, we sought to determine if the RGD sequences in matrix molecules and recognition of these sequences by keratinocytes played a role in the locomotion of keratinocytes. After allowing the cells to attach to the matrix, RGD-containing peptides or control peptides were added to a keratinocyte migration assay. The addition of RGD-containing peptide dramatically inhibited keratinocyte locomotion on a matrix of fibronectin but not on collagen matrices. Therefore, RGD recognition is a critical step for fibronectin-mediated migration but not for collagen-mediated migration. Because the RGD sequences are recognized by cell-surface integrin receptors in a number of cell types, we next examined the roles of integrin receptors in human keratinocyte migration. Using monospecific antibodies that recognize integrin subunits, we found that blocking the beta 1 subunit inhibited the migration of keratinocytes on matrices of fibronectin, interstitial collagen, and basement membrane collagen. Blocking the alpha 5 beta 1 receptor significantly inhibited migration on fibronectin but not on collagen matrices. Conversely, blocking the alpha 2 beta 1 receptor inhibited migration on collagen matrices but not on fibronectin. Blocking the alpha 3 beta 1 receptor uniquely enhanced migration on fibronectin and collagen matrices. In contrast to cells apposed to matrices without the receptor blocked, the enhanced migration in the presence of anti-alpha 3 beta 1 antibody occurred at the later time points of the migration assay. The enhancement of migration by blocking the alpha 3 beta 1 integrin receptor suggests that the interaction of the alpha 3 beta 1 receptor with matrices is associated with immobility.

Amino Acid Sequence↗
PubMed1991

Integrin expression in malignant melanoma.

Invasion of melanoma cells into the underlying interstitial stromal matrix is the initial step for subsequent local and distant metastasis. The invading tumor cell must interact with the extracellular matrix during the early stages of invasion and later during penetration of lymphatic and blood vessels. This interaction with different types of extracellular matrix predicts that the invasive cell must possess surface adhesion receptors with diverse ligand specificities, including the capacity to bind different types of collagens and adhesive glycoproteins. Metastatic melanoma cells do in fact express multiple adhesion receptors, including several of the receptors from the integrin family of heterodimers. The integrin receptors can be either extremely specific for a single ligand or capable of binding multiple ligands. It is likely that the tumor cell's repertoire of adhesion receptors may influence not only its adhesive properties but its metastatic characteristics as well. There is evidence that normal melanocytes have an integrin profile distinct from that of melanoma cells. In particular, melanocytes adhere poorly to laminin while metastatic melanoma cells bind well to this ligand. This difference in adhesion between the two cell types appears to reflect the fact that melanoma cells express a melanoma-specific integrin (alpha 7 beta 1) that binds laminin and is not detectable in normal melanocytes. The presence of increased laminin receptors and enhanced laminin binding in melanoma cells may contribute to the malignant phenotype.

Animals↗
PubMed1991

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.

Cell Adhesion↗
PubMed1991

Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.

Calcium↗
PubMed1991

Laminin-binding integrin alpha 7 beta 1: functional characterization and expression in normal and malignant melanocytes.

A novel integrin, alpha 7 beta 1, that specifically binds with high affinity to laminin has been identified on melanoma cells. This complex was purified from both human and murine melanoma cells by laminin-affinity chromatography, and the alpha 7 subunit was recovered after gel electrophoresis. N-terminal amino acid sequence analysis of the alpha 7 subunit from both human and mouse cells verifies that this integrin is distinct from other alpha chains in the beta 1 family, although strikingly similar to the alpha 6 subunit. By using specific proteolytically derived fragments of laminin, it was determined that the alpha 7 beta 1 complex binds selectively to the E8 region, which represents part of the long arm of laminin. In contrast, the receptor failed to bind to the P1 fragment, which contains the intersection of the short arms of laminin. Although the alpha 7 beta 1 complex was commonly expressed in melanoma cells, this integrin was not detected in normal melanocytes, suggesting that alpha 7 expression may be associated with malignant transformation. These results establish the existence of a novel integrin that binds to the E8 domain of laminin and appears to mediate cell adhesion to this ligand.

Amino Acid Sequence↗
PubMed1991

Role of laminin-binding integrin in the invasion of basement membrane matrices by fibrosarcoma cells.

Laminin is a large glycoprotein that is found in basement membranes and promotes cell adhesion and migration. In human fibrosarcoma cells we detected the presence of an integrin complex, with a Mr of 140,000/120,000 under nonreducing conditions, that bound specifically to laminin-Sepharose columns. Immunoprecipitation with monoclonal antibodies characterized this complex as alpha 6 beta 1. Attachment of the fibrosarcoma cells to laminin substrates was completely inhibited in the presence of anti-alpha 6 beta 1 antibody, while attachment to fibronectin and type IV collagen was unaffected. When seeded onto reconstituted basement membrane, the fibrosarcoma cells spread out, migrated, and invaded the matrix. In the presence of anti-beta 1 or anti-alpha 6 beta 1 antibodies, initial invasion through the matrix was inhibited. The results indicate that the HT1080 cells express the alpha 6 beta 1 complex and that it mediates their attachment to laminin. Furthermore, this receptor appears to be important during initial attachment and subsequent invasion of basement membrane-like matrices.

Amino Acid Sequence↗
PubMed1990

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration.

Calcium channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca2+ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca2+ and K+ channels. Ca2(+)-activated K+ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca2+ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihydropyridine-sensitive Ca2+ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca2+ channels. The modulation of Ca2(+)-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.

Calcium↗
PubMed1990

Analysis of integrin receptors for laminin and type IV collagen on metastatic B16 melanoma cells.

As tumor cells invade surrounding tissue, they adhere to various extracellular matrix components. Previously we reported that B16-BL6 melanoma cell adhesion to both basement membrane and purified protein substrates was blocked by antibody to beta 1-integrin adhesion receptors (R. H. Kramer et al., Cancer Res., 49: 393-402, 1989). In the present study we found, using immunofluorescent staining, that beta 1-integrin complexes were colocalized with vinculin in focal adhesion plaques on laminin, type IV collagen, and fibronectin substrates. To identify potential adhesion receptors on B16 cells, the cells were surface-labeled with 125I, solubilized with detergent, and chromatographed on laminin-, type IV collagen-, and fibronectin-Sepharose columns. On laminin-Sepharose, an integrin heterodimer complex was eluted with EDTA that contained a beta 1 chain at Mr 120,000 and an alpha subunit at Mr 140,000 (nonreduced). This complex was specific for laminin and failed to bind to collagen- or fibronectin-Sepharose columns. Immunoprecipitation with specific monoclonal antibody identified this complex as alpha 6 beta 1 (VLA-6). Furthermore, monoclonal antibody to the alpha 6 beta 1 complex effectively blocked the attachment of B16-BL6 cells to laminin but did not affect adhesion to fibronectin or type IV collagen. We recovered a different integrin complex from type IV collagen-Sepharose columns that was composed of a beta 1 chain and an alpha chain of Mr 180,000 (nonreduced). This same complex also exhibited a weak affinity for laminin-affinity chromatography. The laminin-binding complex and the type IV collagen-binding complex were clearly distinct from the fibronectin-binding receptor and were not eluted by arginyl-glycyl-aspartate-containing peptides. The results suggest that the B16 melanoma cells express multiple integrin-related receptors that appear to mediate cell adhesion to basement membrane matrices.

Animals↗
PubMed1990

Patch cramming: monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels.

This paper introduces "patch cramming," a new procedure that utilizes an ion channel gated directly by an intracellular messenger molecule as a probe for detecting changes in the concentration of that molecule in an intact cell. A patch pipette containing the channel in a membrane patch is inserted into a recipient cell where the channel locally "senses" the intracellular messenger. In this study patches containing Ca2(+)-dependent K+ channels were inserted into Helix neurons, where they were activated by Ca2+ influx during trains of action potentials. Channels gated directly by other messengers, including cyclic nucleotides and IP3, have also been identified. Hence, by using detector channels with appropriate specificity, it may be possible to detect local intracellular fluctuations of these molecules.

Animals↗
PubMed1990

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin.

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.

Antibodies, Monoclonal↗
PubMed1990

Activity-dependent neuromodulation in Aplysia neuron R15: intracellular calcium antagonizes neurotransmitter responses mediated by cAMP.

1. The effect of electrical activity on the response to the neuromodulators serotonin (5-HT) and the neuropeptide egg-laying hormone (ELH) was studied in the Aplysia bursting pacemaker neuron R15. 2. Previous work has shown that 5-HT and ELH augment R15s bursting activity by enhancing two ionic currents, an inwardly rectifying K+ current (IR) and a voltage-gated Ca2+ current (ICa), and that the enhancement of the currents is mediated by the intracellular second-messenger adenosine 3',5'-cyclic monophosphate (cAMP). Here we show that both spontaneous action potentials and voltage-clamp depolarizations suppress the modulation by 5-HT and ELH of these currents. Both spontaneous and evoked depolarizations decrease the magnitude and dramatically speed the decay of the modulation of IR and ICa. 3. The depolarization-induced suppression is blocked by intracellular ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N',-tetraacetic acid (EGTA), indicating that the suppression is Ca-dependent. The suppression is specific for responses mediated by cAMP; a non-cyclic AMP-mediated response to acetylcholine is not affected by depolarizing pulses. 4. The Ca-dependent suppression of IR modulation differs from the Ca-dependent suppression of ICa modulation. Ca2+ influx decreases the sensitivity of IR to neuromodulators without reducing the maximal response elicited by high concentrations of neuromodulators. In contrast, Ca2+ not only decreases the sensitivity of ICa but also reduces the maximal effect elicited by high concentrations of neuromodulators. We have shown previously that intracellular Ca2+ also inactivates the basal IR and ICa in neuron R15 by distinct mechanisms. The inactivation of IR is due to an antagonistic action of Ca2+ on cAMP metabolism, whereas the inactivation of the basal ICa is due primarily to a more direct action of Ca2+, perhaps on the Ca channels themselves. 5. We also studied the interaction between action potentials and neuromodulator released onto R15 from an endogenous source: bag cell neurons, which release large amounts of ELH during an intense "afterdischarge." IR and ICa become greatly enhanced during the afterdischarge, even though R15 continually fires action potentials. In addition, Ca-dependent inactivation of IR is suppressed during the afterdischarge. We suggest that the bag cells release an amount of ELH sufficient to temporarily saturate the cAMP-mediated enhancement of IR and that this temporarily prevents the suppressive effects of Ca2+ on IR. 6. The activity-dependent suppression of neuromodulation in neuron R15 is an example of neuronal plasticity that results from interactions between intracellular messengers.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1990

Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen.

Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells during contraction and in promoting cellular migration after vessel injury. We found that rat aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen types I and IV. Cell attachment to these substrates appears to be mediated by members of the beta 1 integrin family of extracellular matrix receptors. Antibodies to the beta 1 subunit not only demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were recognized by antisera to beta 1 integrin receptors. Each of the receptors appeared to be a heterodimer in which one of several alpha subunits shared a common 120-kDa (nonreduced) beta 1 subunit protein. The rat aortic smooth muscle cells had one alpha subunit (150 kDa nonreduced, 140 kDa reduced) that bound exclusively to fibronectin. There was a second alpha subunit (150 kDa nonreduced, 160 kDa reduced) that bound exclusively to collagen type I. In addition, there was a third alpha subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to collagen types I and IV as well as to laminin; the 185-kDa alpha subunit appeared to bind to collagen more efficiently than it did to laminin. Thus, smooth muscle cells express multiple integrin receptors with different ligand specificities that appear to mediate cell interactions with the extracellular matrix.

Animals↗
PubMed1989

Human melanoma cells express a novel integrin receptor for laminin.

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.

Antibodies, Monoclonal↗
PubMed1989

Identification of integrin collagen receptors on human melanoma cells.

Integrin receptors may mediate the adhesion of cells to a number of extracellular matrix components. We found that the attachment of human melanoma cells to collagen types I and IV was blocked by antibodies to the integrin beta 1 subunit but not by peptides containing the Arg-Gly-Asp sequence. Ligand affinity chromatography was used to search for integrin-related receptors which mediate adhesion to native collagens. Detergent extracts of surface 125I-iodinated melanoma cells were chromatographed on type I or IV collagen-Sepharose columns. Bound material was eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. EDTA, but not Arg-Gly-Asp peptides, eluted a mixture of two integrin-related heterodimeric complexes. Each complex contained the integrin beta 1 chain with Mr of 110,000 and a distinct alpha chain with Mr of either 200,000 or 150,000. Immunoprecipitation with specific monoclonal antibodies identified the complexes as very late activation antigen (VLA)-1 (alpha 1 beta 1) and VLA-2 (alpha 2 beta 1), respectively. The binding of these receptors to collagen appeared to be specific because they failed to be retained on fibronectin- or laminin-Sepharose columns. Immunofluorescent staining of cells on collagen substrates with antibodies to VLA-1 and VLA-2 localized these complexes in vinculin-positive adhesion plaques. In contrast, the receptor complexes were not detected in adhesion plaques of cells attached to fibronectin- or laminin-coated substrates. These results indicate that melanoma cells express at least two different integrin-related collagen-binding receptor complexes that appear to mediate cell adhesion to collagen.

Antibodies↗
PubMed1989

Melanoma cell adhesion to basement membrane mediated by integrin-related complexes.

During invasion and metastasis, tumor cells use a variety of surface adhesion receptors to attach to and invade basement membranes and interstitial stroma. We examined the role of the cell surface integrin-like complex in the attachment of the invasive murine B16-BL6 melanoma cell line to basement membrane. Polyclonal antibodies prepared against integrin-related complexes isolated from hamster BHK cells (anti-ECMR) or mouse erythroleukemia cells (anti-mouse FnR) inhibited the attachment of B16 cells to complex basement membrane matrices and to substrates coated with purified extracellular matrix components (fibronectin, laminin, and type IV collagen). The expression of integrin-like receptors on the surface of B16 cells was confirmed by selective immunoprecipitation of radiolabeled and solubilized membrane proteins with the antibodies. Both antibodies also reacted with an integrin-related fibronectin-binding receptor complex purified by ligand affinity chromatography on fibronectin-Sepharose columns. The anti-integrin antibodies failed to react with the Mr 68,000 laminin-binding protein, suggesting that their inhibition of cell attachment to laminin and complex basement membrane was not due to contaminating antibodies against the Mr 68,000 laminin receptor. The results indicate that the integrin receptor complexes on B16-BL6 cells either interact directly with a diverse set of extracellular-matrix-associated components or somehow modulate the activity and function of other receptors. Thus integrins may have an important role in tumor cell invasion of tissue barriers.

Animals↗