Oral octreotide absorption in human subjects: comparable pharmacokinetics to parenteral octreotide and effective growth hormone suppression.

CONTEXT: Oral administration of a novel octreotide formulation enabled its absorption to the systemic circulation, exhibiting blood concentrations comparable to those observed with injected octreotide and maintaining its biological activity. OBJECTIVES: The aim of the study was to determine oral octreotide absorption and effects on pituitary GH secretion compared to sc octreotide injection. DESIGN: Four single-dose studies were conducted in 75 healthy volunteers. INTERVENTION: Oral doses of 3, 10, or 20 mg octreotide and a single sc injection of 100 µg octreotide were administered. MAIN OUTCOME MEASURE: We measured the pharmacokinetic profile of orally administrated octreotide and the effect of octreotide on basal and stimulated GH secretion. RESULTS: Both oral and sc treatments were well tolerated. Oral octreotide absorption to the circulation was apparent within 1 h after dose administration. Escalating oral octreotide doses resulted in dose-dependent increased plasma octreotide concentrations, with an observed rate of plasma decay similar to parenteral administration. Both 20 mg oral octreotide and injection of 0.1 mg sc octreotide resulted in equivalent pharmacokinetic parameters [mean peak plasma concentration, 3.77 ± 0.25 vs. 3.97 ± 0.19 ng/ml; mean area under the curve, 16.2 ± 1.25 vs. 12.1 ± 0.45 h × ng/ml); and median time ≥ 0.5 ng/ml, 7.67 vs. 5.88 h, respectively). A single dose of 20 mg oral octreotide resulted in basal (P < 0.05) and GHRH-stimulated (P < 0.001) mean GH levels suppressed by 49 and 80%, respectively. CONCLUSIONS: The results support an oral octreotide alternative to parenteral octreotide treatment for patients with acromegaly.

Expression of small heat shock protein alphaB-crystallin is induced after hepatic stellate cell activation.

Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat alphaB-crystallin. Northern blots confirmed expression of alphaB-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of alphaB-crystallin protein in activated but not quiescent HSCs. alphaB-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of alphaB-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of alphaB-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of alphaB-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, alphaB-crystallin represents an early marker for HSC activation.

Nuclear factor kappaB in proliferation, activation, and apoptosis in rat hepatic stellate cells.

BACKGROUND/AIMS: Activation of the transcription factor NFkappaB has been demonstrated in activated hepatic stellate cells (HSCs). We investigated the role of NFkappaB in proliferation, in activation, and in TNFalpha-induced apoptosis of HSCs. METHODS: NFkappaB activation was inhibited using an adenovirus expressing an IkappaB dominant negative protein (Ad5IkappaB) in both quiescent and activated HSCs. Quiescent HSCs were infected with Ad5IkappaB or an adenovirus expressing beta-galactosidase (Ad5LacZ). The cells were cultured for 7 days. HSCs activation was determined by cell morphology, smooth muscle alpha-actin (alpha-sma) expression, and steady-state mRNA levels of alpha1(I) collagen as assessed by Western blot and RNase protection assay, respectively. Proliferation was determined in culture-activated HSCs by 3H-thymidine incorporation and direct cell counting. Apoptosis was analyzed by infecting quiescent or activated HSCs with Ad5IkappaB or Ad5LacZ, and then treating with TNFalpha. Apoptosis was demonstrated by determining cell number, assessing nuclear morphology, TUNEL assay and caspase 3 activity. RESULTS: After 7 days in culture no differences were noted between the Ad5IkappaB- and the Ad5LacZ-infected cells in the morphology, alpha-sma expression or in alpha1(I) collagen mRNA levels. Ad5IkappaB infection did not modify proliferation in activated HSCs. TNFalpha induced apoptosis only in Ad5IkappaB-infected activated, but not quiescent HSCs. Apoptosis was initially demonstrated 12 h after exposure to TNFalpha. Twenty-four h after the TNFalpha treatment, 60% of the activated HSCs were apoptotic. CONCLUSION: NFkappaB activity is not required for proliferation or activation of HSCs; however, NFkappaB protects activated HSCs against TNFalpha-induced apoptosis.

Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice.

The cytoskeleton of the cardiomyocyte has been shown to modulate ion channel function. Cytoskeletal disruption in vitro alters Na(+) channel kinetics, producing a late Na(+) current that can prolong repolarization. This study describes the properties of the cardiac Na(+) channel and cardiac repolarization in neonatal mice lacking ankyrin(B), a cytoskeletal "adaptor" protein. Using whole-cell voltage clamp techniques, I(Na) density was lower in ankyrin(B)(-/-) ventricular myocytes than in wild-type (WT) myocytes (-307+/-26 versus -444+/-39 pA/pF, P<0.01). Ankyrin(B)(-/-) myocytes exhibited a hyperpolarizing shift in activation and inactivation kinetics compared with WT. Slower recovery from inactivation contributed to the negative shift in steady-state inactivation in ankyrin(B)(-/-). Single Na(+) channel mean open time was longer in ankyrin(B)(-/-) versus WT at test potentials (V(t)) of -40 mV (1.0+/-0.1 versus 0. 61+/-0.04 ms, P<0.05) and -50 mV (0.8+/-0.1 versus 0.39+/-0.05 ms, P<0.05). Ankyrin(B)(-/-) exhibited late single-channel openings at V(t) -40 and -50 mV, which were not seen in WT. Late I(Na) contributed to longer action potential durations measured at 90% repolarization (APD(90)) at 1 Hz stimulation in ankyrin(B)(-/-) compared with WT (354+/-26 versus 274+/-22 ms, P<0.05). From ECG recordings of neonatal mice, heart rates were slower in ankyrin(B)(-/-) than in WT (380+/-14 versus 434+/-13 bpm, P<0.01). Although the QT interval was similar in ankyrin(B)(-/-) and WT at physiological heart rates, QT-interval prolongation in response to heart rate deceleration was greater in ankyrin(B)(-/-). In conclusion, Na(+) channels in ankyrin(B)(-/-) display reduced I(Na) density and abnormal kinetics at the whole-cell and single-channel level that contribute to prolonged APD(90) and abnormal QT-rate adaptation.

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins.

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.

Beta-adrenergic agonists regulate cell membrane fluctuations of human erythrocytes.

1. Mechanical fluctuations of the cell membrane (CMFs) in human erythrocytes reflect the bending deformability of the membrane-skeleton complex. These fluctuations were monitored by time-dependent light scattering from a small area ( approximately 0. 25 microm2) of the cell surface by a method based on point dark field microscopy. 2. Exposure of red blood cells (RBCs) to adrenaline (epinephrine) and isoproterenol (isoprenaline) resulted in up to a 45 % increase in the maximal fluctuation amplitude and up to a 35 % increase in the half-width of the amplitude distribution. The power spectra of membrane fluctuations of control and treated cells revealed that adrenaline stimulated only the low frequency component (0.3-3 Hz). Analysis of the dose-response curves of beta-adrenergic agonists yielded an EC50 of 5 x 10-9 and 1 x 10-11 M for adrenaline and isoproterenol, respectively. Propranolol had an inhibitory effect on the stimulatory effect of isoproterenol. These findings show a potency order of propranolol > isoproterenol > adrenaline. 3. The stimulatory effect of adrenaline was a temporal one, reaching its maximal level after 20-30 min but being abolished after 60 min. However, in the presence of 3-isobutyl-1-methylxanthine, a partial stimulatory effect was maintained even after 60 min. Pentoxifylline and 8-bromo-cAMP elevated CMFs. However, exposure of ATP-depleted erythrocytes to adrenaline or 8-bromo-cAMP did not yield any elevation in CMFs. These findings suggest that the beta-agonist effect on CMFs is transduced via a cAMP-dependent pathway. 4. Deoxygenation decreased CMFs and filterability of erythrocytes by approximately 30 %. The stimulatory effect of isoproterenol on CMFs was 2.2-fold higher in deoxygenated RBCs than in oxygenated cells. 5. Exposure of RBCs to adrenaline resulted in a concentration-dependent increase in RBC filterability, demonstrating a linear relationship between CMFs and filterability, under the same exposure conditions to adrenaline. These findings suggest that beta-adrenergic agonists may improve passage of erythrocytes through microvasculature, enhancing oxygen delivery to tissues, especially under situations of reduced oxygen tension for periods longer than 20 min.

Mechanical fluctuations of the membrane-skeleton are dependent on F-actin ATPase in human erythrocytes.

Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend, to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733-737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent Kd of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATPgammaS) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I), resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.

The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation.

Cell-cell recognition and patterning of cell contacts have a critical role in mediating reversible assembly of a variety of transcellular complexes in the nervous system. This study provides evidence for regulation of cell interactions through modulation of ankyrin binding to neurofascin, a member of the L1CAM family of nervous system cell adhesion molecules. The phosphorylation state of the conserved FIGQY tyrosine in the cytoplasmic domain of neurofascin regulates ankyrin binding and governs neurofascin-dependent cell aggregation as well as cell sorting when neurofascin is expressed in neuroblastoma cells. These findings suggest a general mechanism for the patterning of cell contact based on external signals that regulate tyrosine phosphorylation of L1CAM members and modulate their binding to ankyrin.

Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

Cell membrane fluctuations are regulated by medium macroviscosity: evidence for a metabolic driving force.

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.

Oxygenation-deoxygenation cycle of erythrocytes modulates submicron cell membrane fluctuations.

Low frequency submicron fluctuations of the cell membrane were recently shown to be characteristic for different cell types, nevertheless their physiological role is yet unknown. Point dark-field microscopy based recordings of these local displacements of cell membrane in human erythrocytes, subjected to cyclic oxygenation and deoxygenation, reveals a reversible decrease of displacement amplitudes from 290 +/- 49 to 160 +/- 32 nm, respectively. A higher rate of RBC adhesion to a glass substratum is observed upon deoxygenation, probably due to a low level of fluctuation amplitudes. The variation in the amplitude of these displacements were reconstituted in open RBC ghosts by perfusing them with composite solutions of 2,3 diphosphoglycerate, Mg+2, and MgATP, which mimic the intracellular metabolite concentrations in oxygenated and deoxygenated erythrocytes. The mere change in intracellular Mg+2 during oxygenation-deoxygenation cycle is sufficient to explain these findings. The results imply that the magnitude of fluctuations amplitude is directly connected with cell deformability. This study suggests that the physiological cycle of oxygenation-deoxygenation provides a dynamic control of the bending deformability and adhesiveness characteristics of the RBC via a Mg+2-dependent reversible assembly of membrane-skeleton proteins. The existing coupling between oxygenation-deoxygenation of the RBC and its mechanical properties is expected to play a key role in blood microcirculation and may constitute an example of a general situation for other circulating blood cells, where the metabolic control of cytoskeleton dynamics may modulate their dynamic mechanical properties.

Correlation between local cell membrane displacements and filterability of human red blood cells.

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg(2+)-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180-675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.

A genetic study of Behçet disease in Israel.

The frequency of HLA-B5 in 24 Israeli Behçet disease (BD) patients from various subpopulations was significantly greater than in 615 control individuals (P less than 0.003). The relative risk for a B5 carrier to develop BD was calculated to be 5.0. Six patients were offspring of consanguineous marriages, which is not unexpected in the populations studied. Five of the patients had only one HLA-B antigen, which in four cases was B5. Two of the latter were B5 homozygotes, indicating a possible greater susceptibility. This study confirms the reported association between BD and HLA-B5 in a populationnot previously investigated. Furthermore, these data support the suggestion that B5 is associated with BD in populations deriving from the Mediterranean Basin, the Middle East and Far East.