Klaus Hierholzer (1929-2007) and his impact on our understanding of the renal effects of steroid hormones.

Klaus Hierholzer (1929-2007) dissected various functions influenced by steroids in the distal tubule and showed that aldosterone in low doses reversed the sodium and potassium transport defect in adrenalectomized rats, through a rapid activation of Na+,K+-ATPase. Subsequent studies addressed the role of 11-beta-hydroxysteroid oxidoreductase (11-HSD) and showed that the undisturbed functioning of 11-HSD is a prerequisite for selective mineralocorticosteroid regulation of epithelial transport. Another set of original experiments showed that 11-HSD was equally important in the distal colon, thus establishing that the large intestine acts in parallel with the distal nephron. Hierholzer, born in Konstanz on June 8, 1929, was laureated in medicine on May 25, 1954. Subsequently he worked at the Department of Pharmacology of the University of Freiburg, Cornell University with J. F. Pitts, the Department of Medicine of the University of Frankfurt-am-Main, the University of Copenhagen with H. H. Ussing, and the Institute of Physiology of the Freie Universitaet in Berlin where he became full professor and head of the Institute of Clinical Physiology in 1968. He held that position until 1998. He died in Allensbach in the family house on February 27, 2007. Hierholzer was a member of the Naturforscher Leopoldina Academy and of many other scientific societies, including the Academy of Science and Technology in Berlin, and received various awards including an honorary professorship at the University of Naples, the Bezold Medal, the Volhard Medal, the Schoeller/Junkman Award, and the Malpighi Medal (in memoriam). He published nearly 300 papers including various seminal books. Noteworthy also are his papers on the history of physiology of the kidney and acid-base balance. A total of 26 scientists who trained in his laboratory became professors.

Effects of unoprostone and endothelin 1 on L-type channel currents in human trabecular meshwork cells.

BACKGROUND: The trabecular meshwork (TM) is a smooth muscle-like tissue with contractile properties and by this mechanisms involved in the regulation of aqueous humor outflow. Isopropyl unoprostone (Rescula, Novartis Ophthalmics), a synthetic docosanoid, reduces intraocular pressure in glaucoma patients and normal subjects. In isolated TM strips, unoprostone reduces TM contractility in the presence of endothelin 1 (ET-1). However, the signal transduction pathway of unoprostone still remains unclear. Since L-type channel currents are known to influence the contractility of TM, we examined the effects of unoprostone and ET-1 on L-type channel currents of TM cells. METHODS: The effects of unoprostone, ET-1 and the tyrosine kinase inhibitor herbimycin A on L-type channel currents of cultured human TM cells were investigated using the perforated patch configuration of the patch-clamp technique. RESULTS: Application of ET-1 had no effect on L-type channel currents. Unoprostone led to a dose-dependent reduction of control currents. The effect of unoprostone is independent of ET-1. After preincubation of cells with herbimycin A, unoprostone had no effect on the L-type channel current amplitude. Human TM cells preincubated with herbimycin A showed a reduced current density compared with control cells. Both substances, unoprostone and herbimycin A, increased the inactivation time constant of L-type channel currents. CONCLUSION: We conclude that unoprostone reduces the activity of L-type Ca2+ channels. This effect seems to be independent of ET-1. The signal transduction pathway seems to be mediated by tyrosine kinases.

Effects of ML-7 and Y-27632 on carbachol- and endothelin-1-induced contraction of bovine trabecular meshwork.

The trabecular meshwork is considered a smooth muscle like tissue contributing to aqueous outflow regulation and thus to regulation of intraocular pressure. An elevation in intraocular pressure is one of the greatest risk factors for most forms of glaucoma. We assume that contraction of trabecular meshwork reduces aqueous humor outflow and thus enhances intraocular pressure, whereas relaxation exerts the opposite effect. The present paper supports the hypothesis of the trabecular meshwork being a smooth muscle-like tissue. We perform measurements of isometric force in isolated bovine trabecular meshwork strips. Contractility of this tissue is induced by carbachol or endothelin-1. The contractile force is successfully inhibited by ML-7, a highly specific inhibitor of myosin light chain kinase. The contraction is also reduced in the presence of the RhoA kinase inhibitor Y-27632. We further describe the protein expression of smooth muscle myosin and its regulatory kinase, the myosin light chain kinase, in human and bovine trabecular meshwork cells. Additionally, the serine phosphorylation of myosin light chain kinase is shown. These data indicate that the trabecular meshwork expresses major contractility regulating proteins which are involved in tissue function. Inhibition of the signaling pathways which lead to myosin phosphorylation causes inhibition of contractile force in trabecular meshwork. According to our concept of aqueous humor outflow regulation, trabecular meshwork relaxing substances appear to be ideal antiglaucomatous drugs, leading to increased outflow facility.

Stimulation of cannabinoid (CB1) and prostanoid (EP2) receptors opens BKCa channels and relaxes ocular trabecular meshwork.

Prostanoids and cannabinoids have ocular hypotensive and neuroprotective properties. The effect of the prostanoid AH13205 (EP2), the thromboxane-mimetic U46619, the cannabinoid (CB) agonists WIN55212-2 and CP 55,940, endothelin-1 (ET-1) and 8-bromo-cAMP on the membrane currents of trabecular meshwork (TM) cells were measured using the patch-clamp technique and compared to their effects on TM contractility. Previous studies show relaxation of TM to AH 13205 and other substances that elevate cAMP, while U46619 and endothelin-1 contract TM. This study shows that after contraction (100%) with carbachol (10(-6)m), the CB agonist CP 55,940 dose-dependently reduced contractility to 83+/-4% (n=9) (10(-6)m) and 61+/-10%, (n=7) (10(-5)m). In the presence of both the CB1 antagonist AM251 (10(-6)m) and CP 55,940 (10(-5)m), the contractile response to carbachol reached 84+/-3% (n=6) of the original level. In patch-clamp experiments, membrane permeable 8-bromo-cAMP (10(-4)m) had no effect on currents of TM cells. In contrast, AH 13205 and two cannabinoids reversibly enhanced outward current through high-conductance Ca(2+)-activated K(+) channels (BKCa, BK, maxi-K) to the following values (in % of the initial value at 100 mV): AH 13205 (10(-5)m): 200+/-28% (n=6), CP 55,940 (10(-6)m): 196+/-33% (n=7), CP 55,940 (10(-5)m): 484+/-113% (n=7), WIN55212-2 (10(-5)m): 205+/-41% (n=10). Iberiotoxin (10(-7)m) completely blocked these responses. The current response to CP 55,940 (10(-5)m) could be partially blocked by the CB1 antagonist AM251 (10(-6)m). Conversely, the contractile agents in this study either caused a transient reduction in outward current (ET-1(5x10(-8)m)) or had no effect (U46619 (10(-6)m)). We conclude that stimulation of EP2 and CB1 receptors in TM is coupled to the activation of BKCa channels via a non-diffusible second messenger cascade. This effect may contribute to the relaxant activity of EP2 and CB1 agonists in isolated TM strips, modulating ocular outflow.

Electrophysiological response of cultured trabecular meshwork cells to synthetic ion channels.

The response of living cells of the trabecular meshwork to synthetic ion channels is described. The THF-gramicidin hybrids THF-gram and THF-gram-TBDPS as well as a linked gA-TBDPS and gramicidin A were applied to cultured ocular trabecular meshwork cells. THF-gram application (minimal concentration, 10(-8) M; saturation, 10(-7) M) led to an additional conductance which displayed characteristics of weak Eisenman-I-selective cation channels, no cell destruction, an asymmetric change of the inward/outward currents, and higher current densities using Cs(+) as charge carrier compared to Na(+) and K(+). Linked-gA-TBDPS showed at 10(-12) M increases of the membrane conductance comparable to gA at 10(-7) M and a much faster response of the cells. Thus, THF-gramicidin hybrids form a basis for the use of synthetic ion channels in biological systems, which eventually may lead to new therapeutic approaches.

Effects of protein kinase C on delayed rectifier K+ channel regulation by tyrosine kinase in rat retinal pigment epithelial cells.

PURPOSE: Investigation of the regulation of delayed rectifier potassium channels by protein kinases in retinal pigment epithelial (RPE) cells. METHODS: Membrane currents of cultured RPE cells were measured in either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: Different potassium channel blockers from animal venoms (charybdotoxin, hanatoxin, agitoxin, tityustoxin) were used to identify the delayed rectifying outward current in RPE cells as current through potassium channels, mainly composed of Kv1.3 subunits. Extracellular application of a tyrosine kinase blocker, such as genistein (100 microM), lavendustin A (10 microM), or herbimycin A (4 microM), almost completely abolished the delayed rectifier currents. Intracellular application of the tyrosine kinase pp60(c-src) (30 U/mL) through a patch pipette led to an increase of the delayed rectifier current consistent with an activation of the delayed rectifier channels by src subtype tyrosine kinase. Inhibition (with 1 microM chelerythrine) or activation (with 1 microM phorbol ester PMA) of protein kinase C (PKC) did not change the activity of delayed rectifier channels. Inhibition of PKC also did not change the effect of tyrosine kinase inhibition of delayed rectifier currents. However, in cells with stimulated PKC, inhibition of tyrosine kinase by genistein led to an increase of delayed rectifier currents. Intracellular application of pp60(c-src) (30 U/mL) in cells with stimulated PKC led to inhibition of delayed rectifier currents. CONCLUSIONS. Tyrosine kinase, of the src family, can activate or inhibit delayed rectifier channels composed of Kv1.3 subunits in RPE cells. The activity of PKC determines whether tyrosine kinase activates or inhibits these K+ channels.

Mecanismos de la secreción del humor acuoso y su modulación por drogas / Aqueous humor secretion mechanisms and it modulation by drugs

Recordemos que el epitelio ciliar consta de dos cepas de células, pigmentadas y no pigmentadas; nuestro concepto es que existe un Sincitium funcional (recordemos que el Sincitium se define como citoplasma con varios núcleos sin límites celulares definidos), esto se refiere a que la membrana basal lateral de las células pigmentadas tiene todos los transportadores que necesita para llevar hacia arriba el cloruro de sodio y luego en consecuencia el agua. Esta membrana tiene el importante intercambio de Na + - K + y obviamente tiene un canal de cloruro y también una etapa de salida de bicarbonato. Nosotros pensamos que esta etapa de salida también puede ser modulada por los inhibidores de la anhidrasa carbónica. Recientemente se mostró en imágenes de video que este concepto es verdadero en el epitelio completo donde ambas cepas de células están juntas; se demostró que los electrolitos son llevados hacia arriba, al interior de la célula, desde las pigmentadas hacia las no pigmentadas y en ellas a la etapa de salida. Por lo tanto hemos visto como el mecanismo del humor acuoso es muy complejo, su secreción comprende apareamiento de transportadores y canales entre células pigmentadas y no pigmentadas y las dos capas de células son funcionalmente una, un Sincitium. Es importante el flujo constante del humor acuoso a través de las cámaras del ojo para una función visual normal. Es necesario un globo ocular formado por una presión intraocular adecuada para mantener la eficacia óptica. Además, el humor acuoso aporta los sustratos necesarios para la función metabólica normal de los tejidos oculares avasculares a los cuales bañan particularmente cristalino, córnea y red trabecular; este flujo de humor acuoso se encarga además de remover los desperdicios metabólicos. Es también un medio para que el iris responda a la luz...