Stefan Mergler

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells.

The effect of protein tyrosine kinases (PTKs) on L‐type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L‐type channels were measured in the perforated patch‐clamp technique and identified by using the L‐type calcium channel opener Bay K8644 (10−6 M). Application of the PTK blockers genistein (5 × 10−6 M) or lavendustin A (5×10−6 M) led to a decrease of L‐type currents. The inactive genistein analog daidzein (10−5 M) showed no effect on calcium channels. Intracellular application of pp60c‐src (30 U/ml) via the patch‐pipette during the conventional whole‐cell configuration led to an increase of L‐type currents. The protein kinase A and protein kinase G blocker H9 (10−6 M) showed no effect on L‐type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10−5 M) reduced the L‐type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5×10−5 M) for PKC inhibition led to a fast rundown of L‐type current amplitudes. Intracellularly applied myristoylated PKC substrate (10−4 M) together with pp60c‐src showed no effect on L‐type current. Up‐regulation of PKC by 10−6 M phorbol‐12‐myristate‐13‐acetate (PMA) had no effect on the L‐type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L‐type currents. Intracellular application of pp60c‐src in PMA‐treated cells led to a reduction of L‐type currents. We conclude that in the resting cell, PTK and PKC regulate L‐type calcium channels in an additive manner. L‐type channels appeared as a site of integration of PTK activation and of PKC‐dependent pathways. The activity of PKC determines whether PTK decreases or increases L‐type channel activity.—Strauss, O., Mergler, S., Wiederholt, M. Regulation of L‐type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells. FASEB J. 11, 859–867 (1997)

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringers solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.

The Royal College of Surgeons Rat: An Animal Model for Inherited Retinal Degeneration with a Still Unknown Genetic Defect

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an ‘off’ signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.

Anti-TNF-α Treatment: A Possible Promoter in Endogenous Uveitis? Observational Report on Six Patients: Occurrence of Uveitis Following Etanercept Treatment

Background: TNF-α inhibitory drugs are widely used with beneficial effect in the treatment of rheumatic diseases, such as juvenile idiopathic arthritis and ankylosing spondylitis. Due to the complex immune regulatory function of TNF-α, induction of inflammation in several organs including the eye, skin, and gastrointestinal tract has been reported. This report describes the occurrence of intraocular inflammation after treatment with the TNF-α antagonist etanercept. Methods: In this observational case series, we followed and examined six patients receiving etanercept for juvenile idiopathic arthritis, ankylosing spondylitis, adult Still’s disease, or psoriasis. Result: All patients responded well to their joint affliction, but developed endogenous uveitis for the first time after application of etanercept. Following acute intervention with corticosteroids, etanercept was discontinued and instead an antibody-based anti-TNF treatment using infliximab was instituted. We documented visual acuity before and after change from etanercept to treatment with the anti-TNF-α antibody infliximab. Interestingly, prompt long-term remission (mean 34 months) of uveitis without recurrence could be induced in all patients. Conclusions: Our observations may indicate that immunodysregulatory and even proinflammatory effects of etanercept are of relevance in clinical practice. Further randomized controlled clinical trials are necessary to investigate possible side effects of anti-TNF therapy using etanercept and infliximab.

The human corneal endothelium: New insights into electrophysiology and ion channels

The corneal endothelium is a monolayer that mediates the flux of solutes and water across the posterior corneal surface. Thereby, it plays an essential role to maintain the transparency of the cornea. Unlike the epithelium, the human endothelium is an amitotic cell layer with a critical cell density and the risk of corneal decompensation. The number of endothelial cells subsequently decreases with age. Moreover, the endothelial cell loss is accelerated after various impairments such as surgical trauma (e.g. cataract extraction) and following corneal transplantation. This cell loss is associated with programmed cell death (apoptosis) and changed ion channel activity. However, little is known about the electrophysiology and ion channel expression (in particular Ca2+ channels) in corneal endothelial cells. This article reviews our current knowledge about the electrophysiology of the corneal endothelium. It highlights ion channel expression, which may have a major role in corneal cell physiology and pathological events. A better understanding of the (electro)physiological function of the cornea may lead to the development of clinical relevant new therapeutic and preventive measures.

TRPV channels mediate temperature-sensing in human corneal endothelial cells

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca(2+) transients and induction of these responses through rises in ambient temperature from 25 degrees C to over 40 degrees C. The currents underlying Ca(2+) transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 microM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 degrees C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La(3+)) (100 microM) and ruthenium-red (RuR) (10 microM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 microM) led to a reversible increase in intracellular Ca(2+) indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.

Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration

The effect of protein tyrosine kinases (PTK) on L‐type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10–6M) sensitive L‐type channels were measured using the patch‐clamp technique. The current density of L‐type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5× 10–6 M) led to an increase of L‐type currents. Intracellular application of pp60c‐src (30 U/ml) via the patch pipette led to a transient decrease of L‐type currents. The protein kinase A (PKA) and PKG blocker H9 (10–6 M) showed no effect on L‐type currents. However, the protein kinase C blocker chelerythrine (10–5 M) reduced these currents. Up‐regulation of PKC by 10–6 M 4β‐phorbol‐12 myristate‐13 acetate (PMA) led to a decrease of L‐type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60c‐src in PMA‐treated cells led to a transient increase of L‐type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L‐type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS‐RPE cells, this disturbed regulation of L‐type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.—Mergler, S., Steinhausen, K., Wiederholt, M., Strauss, O. Altered regulation of L‐type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration. FASEB J. 12, 1125–1134 (1998)

Thermosensitive transient receptor potential channels in human corneal epithelial cells

Thermosensitive transient receptor potential (TRP) proteins such as TRPV1–TRPV4 are all heat‐activated non‐selective cation channels that are modestly permeable to Ca2+. TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca 2+]i transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT‐PCR and TRPV2 transcripts were also detected. In fura2‐loaded HCEC, a TRPV1–3 selective agonist, 100 µM 2‐aminoethoxydiphenyl borate (2‐APB), induced intracellular Ca2+ transients and an increase in non‐selective cation outward currents that were suppressed by ruthenium‐red (RuR) (10–20 µM), a non‐selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40°C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La3+) (100 µM) suppressed these temperature‐induced Ca2+ increases. Planar patch‐clamp technique was used to characterize the currents underlying Ca2+ transients. Increasing the temperature to over 40°C induced reversible rises in non‐selective cation currents. Moreover, a hypotonic challenge (25%) increased non‐selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermo‐sensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress. J. Cell. Physiol. 226: 1828–1842, 2011.

Transient Receptor Potential Channel TRPM8 Agonists Stimulate Calcium Influx and Neurotensin Secretion in Neuroendocrine Tumor Cells

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca2+]i). Our previous study demonstrated that Ca2+-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87–102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca2+]i and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca2+]i in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca2+ transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca2+]i and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.

Capsaicin induces cytotoxicity in pancreatic neuroendocrine tumor cells via mitochondrial action.

Capsaicin (CAP), the pungent ingredient of chili peppers, inhibits growth of various solid cancers via TRPV1 as well as TRPV1-independent mechanisms. Recently, we showed that TRPV1 regulates intracellular calcium level and chromogranin A secretion in pancreatic neuroendocrine tumor (NET) cells. In the present study, we characterize the role of the TRPV1 agonist - CAP - in controlling proliferation and apoptosis of pancreatic BON and QGP-1 NET cells. We demonstrate that CAP reduces viability and proliferation, and stimulates apoptotic death of NET cells. CAP causes mitochondrial membrane potential loss, inhibits ATP synthesis and reduces mitochondrial Bcl-2 protein production. In addition, CAP increases cytochrome c and cleaved caspase 3 levels in cytoplasm. CAP reduces reactive oxygen species (ROS) generation. The antioxidant N-acetyl-l-cysteine (NAC) acts synergistically with CAP to reduce ROS generation, without affecting CAP-induced toxicity. TRPV1 protein reduction by 75% reduction fails to attenuate CAP-induced cytotoxicity. In summary, these results suggest that CAP induces cytotoxicity by disturbing mitochondrial potential, and inhibits ATP synthesis in NET cells. Stimulation of ROS generation by CAP appears to be a secondary effect, not related to CAP-induced cytotoxicity. These results justify further evaluation of CAP in modulating pancreatic NETs in vivo.