The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration.

1. Brain capillary endothelial cells express a variety of nucleotide receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype. 2. Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca(2+)](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased [Ca(2+)](i) to similar levels, while 2-MeSADP, ADP and adenosine gave smaller responses. 3. Removal of extracellular calcium caused no significant change in the [Ca(2+)](i) response to 2-MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor. 4. All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% responded to adenosine and 50% to 2-MeSADP. No cells responded to alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca(2+)](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca(2+)](i) response to the P2Y(1)-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS). 6. The P2Y(2) antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7. The A(1) subtype-selective adenosine receptor agonist N(6)-Cyclopentyladenosine (CPA) gave a small but characteristic [Ca(2+)](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca(2+)](i) changes. 8. The results are consistent with the presence on RBCEC of a P2Y(2)-like receptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing intracellular Ca(2+). The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.

Identification of thrombin receptors in rat brain capillary endothelial cells.

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.

Na+-Ca2+ exchange and its implications for calcium homeostasis in primary cultured rat brain microvascular endothelial cells.

1. The role of Na+-Ca2+ exchange in the regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was studied in primary cultured rat brain capillary endothelial cells. [Ca2+]i was measured by digital fluorescence imaging in cells loaded with fura-2. 2. ATP (100 microM) applied for a short period of time (6 s) caused a rise in [Ca2+]i from 127 +/- 3 (n = 290) to 797 +/- 25 nM, which then declined to the resting level, with a t time required for [Ca2+]i to decline to half of peak [Ca2+]i) of 5.4 +/- 0.09 s. This effect was independent of external Ca2+ and could be abolished by previously discharging the Ca2+ pool of the endoplasmic reticulum with thapsigargin (1 microM). 3. Application of thapsigargin (1 microM) or cyclopiazonic acid (10 microM) to inhibit the Ca2+-ATPase of the endoplasmic reticulum 6 s prior to ATP application did not influence the peak [Ca2+]i but greatly reduced the rate of decline of [Ca2+]i, with t values of 15 +/- 1.6 and 23 +/- 3 s, respectively. 4. In the absence of external Na+ (Na+ replaced by Li+ or N-methylglucamine) the basal [Ca2+]i was slightly elevated (152 +/- 6 nM) and the restoration of [Ca2+]i after the ATP stimulation was significantly slower (t , 7.3 +/- 0.46 s in Li+ medium, 8.12 +/- 0.4 s in N-methylglucamine medium). 5. The external Na+-dependent component of the [Ca2+]i sequestration was also demonstrated in cells stimulated by ATP subsequent to addition of cyclopiazonic acid; in a Na+-free medium [Ca2+]i remained at the peak level in 88 % of the cells after stimulation with ATP. 6. Addition of monensin (10 microM) in the presence of external Na+ increased the resting [Ca2+]i to 222 +/- 9 nM over approximately 1 min and subsequent removal of extracellular sodium resulted in a further increase in [Ca2+]i to a peak of 328 +/- 11 nM, which was entirely dependent on external Ca2+. 7. These findings indicate that a functional Na+-Ca2+ exchanger is present at the blood-brain barrier, which plays a significant role in shaping the stimulation-evoked [Ca2+]i signal and is able to work in reverse mode under pharmacological conditions.

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix.

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.

[Surgery of infected hand injuries in Bács-Kiskun County]. / A septicus kézsebészet fejlödése Bács-Kiskun megyében.

The therapy of the diseases of the infected hand belongs to the most difficult tasks of the hand surgery. In the county Bács-Kiskun we have reached important results during the last two decades, described in this paper, together with the Hand Surgery Work Groups of the Accident Departments of Kecskemét (County Hospital), Baja and Kiskunhalas.

[Critical analysis of osteosynthesis of fractures of the femoral diaphysis]. / Femur diaphysis törések osteosynthesiseinek kritikai elemzése.

Authors analyse the osteosyntheses of 98,417 segmental fractures of the femoral diaphysis. Beside the 72 per cent of excellent and good results in 19 per cent complications were found, especially after using plate osteosynthesis. In spite of the fact that Kuentscher nailing seems to be the method of choice in this paper the problem is not analysed from the viewpoint of indication but from that of the causes of prolonged healing. On the basis of their own experiences they call attention to the danger of complications. They think the choice of the most suitable method of treatment of very great importance and also the regular control, the aftertreatment and readiness to cooperation of the patients.

Intensive postoperative mydeton therapy of traumatological patients.

The effect of high-dose oral Mydeton therapy has been examined during the postoperative treatment of 73 patients who had undergone traumatological operations. By controlling the most characteristic parameters it has been observed that due to its myotonolytic and lymph flow improving action Mydeton improves significantly motor function and articular movement, shortens the postoperative therapeutic period, and facilitates the performance of curative physical exercise. Since the drug is well tolerated by the patients its use may be recommended as a routine in the above mentioned traumatological indications.

[The primary operative treatment of the ligament lesions following to cubital dislocation (author's transl)]. / A könyökízÿlet ficam utáni szalagsérüléseinek primer mütéti ellátása.

The operative treatment of ligament lesions subsequent to instable cubital dislocations have been suggested. According to the authors observations, the exposure of the cubital dislocations with relapsing tendencies and the suture adaptations of ruptured collateral ligaments are more successful than the conservative treatment.

[Internal fixation for fractures of the lateral malleolus with titanium "crown"-plates (author's transl)]. / A külbokatörések osteosynthesise titán koronalemezzel.

This team of authors constructed a so called "crown"-plate from titanium, suitable for fixation of lateral malleolar fractures. The plate seems to be ideal for Weber-B type fractures, which are level with the joint space, and it can be applied in the more distal (Weber-A) and proximal (Weber-C) fractures too. The titanium plate was used in 166 fractures with good results. On the basis of the follow up assessment of 151 healed cases the authors feel encouraged to suggest the use of the titanium crown plate for internal fixation of lateral malleolar fractures.

[Operative treatment of acute acromioclavicular dislocation (author's transl)]. / A friss acromioclavicularis ficam mütéi kezelése.

Author describes clinical appearance of the acromioclavicular dislocation. Discussing different surgical techniques, a new combination of surgical procedures is recommended. In case of acute acromioclavicular dislocation beside the osteosynthesis by means of traction wire loop, the reconstruction of the coracoclavicular ligament and the fixation of clavicula with tendon graft should be performed. Good results were obtained with the above method.

[Ligamentous injuries of the wrist joint (author's transl)]. / A csuklóízület szalagsérülései.

Author deal with some basic type of ligamentous injuries around the wrist. Attention is drown to the significance of injuries to the distal radio-ulnar ligamentsystem, the triangular fibrocartilage, and ulnar carpal ligament. Diagnostic and therapeutic methods are recommended. The importance for treatment of ligamentous injuries around the wrist joint is emphasized.