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Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K(+) channels (K(ATP) channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channel-activating effect of K(ATP) channel openers such as the pinacidil analog P1075. We investigated the inhibition by MgATP and P1075 of glibenclamide binding to SUR2B, the SUR subtype in smooth muscle. To increase specific binding, experiments were also performed using SUR2B(Y1206S), a mutant with higher affinity for glibenclamide than for the wild-type (K(D )= 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. In the absence of MgATP, [(3)H]glibenclamide binding to both SURs was homogenous. MgATP inhibited [(3)H]glibenclamide binding to both SURs to 25% by reducing the apparent number of glibenclamide binding sites, leaving the affinity unchanged. In the absence of MgATP, P1075 inhibited [(3)H]glibenclamide binding in a monophasic manner with K(i) approximately 1 microM. In the presence of MgATP (1 mM), inhibition was biphasic with one K(i) value resembling the true affinity of P1075 for SUR2B (2-6 nM) and the other resembling K(i) in the absence of MgATP (approximately 1 microM). The data show that (1) MgATP induces heterogeneity in the glibenclamide sites; (2) the high-affinity glibenclamide sites remaining with MgATP are linked to two classes of P1075 sites; and (3) P1075 interacts specifically with SUR2B also in the absence of MgATP. The data are discussed with the assumption that SUR2B, expressed alone, forms tetramers; that MgATP induces allosteric interactions between the subunits; and that mixed SUR2B-glibenclamide-P1075 complexes can exist at equilibrium.

Background: The success of intravascular brachytherapy relies entirely on the interdisciplinary approach. Interventional cardiologists, radiation oncologists and medical physicists must form a team from day 1. All members of the team need special knowledge and regular training in the field of vascular radiation therapy. Optimization of intravascular brachytherapy requires the use of standardized methods of dose specification, recording and reporting. This also implies using standardized methods of source calibration in terms of absorbed dose to water and having methods for simple internal control of the dosimetric quantities of new or replaced sources. Guidance is offered by international recommendations (AAPM TG 60, DGMP Report 16, NCS and EVA GEC-ESTRO). LEGAL REQUIREMENTS FOR RADIATION PROTECTION--WHAT'S NEW?: In Europe, new legal requirements on radiation protection issues have to be fulfilled. For Germany, the revised "Strahlenschutzverordnung" has been released recently. Nearly all organizational and medical processes are affected. For intravascular brachytherapy, several changes of requirements have to be considered. However, to follow these requirements does not cause serious problems. DGMP REPORT 16: GUIDELINES FOR MEDICAL PHYSICAL ASPECTS OF INTRAVASCULAR BRACHYTHERAPY: Evaluation of clinical results by comparison of intravascular brachytherapy treatment parameters is possible only if the prescribed dose and the applied dose distribution are reported clearly, completely and uniformly. The DGMP guidelines thus recommend to prescribe the dose to water at the system related reference point PRef at 2 mm radial distance for intracoronary application (and at 5 mm for peripheral vessels). The mean dose at 1 mm tissue depth (respectively at 2 mm) should be reported in addition. To safely define the planning target volume from the injured length, safety margins of at least 5 mm (10 mm) have to be taken into account on both ends. Safety margins have also to be considered for multisegmental treatment, to omit underdosage. IVUS based localization will support precise planning, avoid a geographic miss and edge effects and will allow for later evaluation. These DGMP recommendations are also included in the EVA GEC ESTRO recommendations and in the draft for an up-date of the AAPM TG 60 report., Conclusion: Medical physical quality management of intravascular brachytherapy is a necessary condition for optimal and safe treatment. Procedures, devices, and sources should fulfill the same degree of precision and safety as common in radiotherapy.

In-stent restenosis has been shown to be associated with a high recurrence rate of repetitive restenosis and remains a challenging task in interventional cardiology. Randomized, placebo-controlled trials have established that beta- as well as gamma-based vascular brachytherapy reduces the incidence of restenosis and clinical event rates following percutaneous coronary intervention (PCI) for the treatment of in-stent restenosis with focal and moderate length. Despite the number of clinical trials with impressive and convincing data, dose finding in most trials is empirical and remains an open question in this fairly new field of percutaneous interventional procedures. Current clinical trials have unequivocally demonstrated a clear dose dependency for the inhibition of intimal proliferation and a significant effectiveness for the treatment of in-stent restenosis with a dose around 20 Gy. Theoretical considerations and empirical data, however, support the need for a dose escalation with current systems to even further improve clinical results. A controlled dose escalation seems, thus, justified and is apparently not related with an increased risk of major adverse cardiac events. The current article gives an overview about theoretical considerations of dosing for intracoronary brachytherapy, presents recent data from important clinical trials in different views, and opens new perspectives for the successful treatment of in-stent restenosis.

The novel sulfonylthiourea 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (HMR 1883), a blocker of ATP-sensitive K(+) channels (K(ATP) channels), has potential against ischemia-induced arrhythmias. Here, the interaction of HMR 1883 with sulfonylurea receptor (SUR) subtypes and recombinant K(ATP) channels is compared with that of the standard sulfonylurea, glibenclamide, in radioligand receptor binding and electrophysiological experiments. HMR 1883 and glibenclamide inhibited [(3)H]glibenclamide binding to SUR1 with K(i) values of 63 microM and 1.5 nM, and [(3)H]opener binding to SUR2A/2B with K(i) values of 14/44 microM and 0.5/2.8 microM, respectively (values at 1 mM MgATP). The interaction of HMR 1883 with the SUR2 subtypes was more sensitive to inhibition by MgATP and MgADP than that of glibenclamide. In inside-out patches and in the absence of nucleotides, HMR 1883 inhibited the recombinant K(ATP) channels from heart (Kir6.2/SUR2A) and nonvascular smooth muscle (Kir6.2/SUR2B) with IC(50) values of 0.38 and 1.2 microM, respectively; glibenclamide did not discriminate between these channels (IC(50) approximately 0.026 microM). In whole cells, the recombinant vascular K(ATP) channel, Kir6.1/SUR2B, was inhibited by HMR 1883 and glibenclamide with IC(50) values of 5.3 and 0.043 microM, respectively. The data show that the sulfonylthiourea exhibits a selectivity profile quite different from that of glibenclamide with a major loss of affinity toward SUR1 and slight preference for SUR2A. The stronger inhibition by nucleotides of HMR 1883 binding to SUR2 (as compared with glibenclamide) makes the sulfonylthiourea an interesting tool for further investigation.

Only those strains of corynebacteria that carry the gene for diphtheria toxin may cause diphtheria. The only known reservoir of C. diphtheriae is man. The past decade has seen a return of diphtheria in the newly independent states of the former Soviet Union. Owing to a lack of immunization of the population, more than 150,000 people went down with the disease. In Germany, too, contact resulted in a number of cases and two deaths. The sole effective protection is immunization. Although more than 90% of children and adolescents are protected, only 40% to 60% of adults are.

Background: Intracoronary devices such as stents or guide wires may disturb the dose distribution of beta sources in cardiovascular brachytherapy. As clinical observations indicate that underdosage increases the risk of restenosis, accurate measurements are mandatory to investigate these effects., Methods and Results: Dose perturbation effects of different interventional equipment were systematically determined. Dose distributions of 90Sr-beta line sources were measured by means of a special set-up employing plastic scintillator dosimeters in a water phantom. Shielding effects were found to be 2-5% for single stents and 5-10% for graft stents, stent-in-stent geometries, and guiding catheters. Guide wires close to the source reduced the dose by 25-30%., Conclusions: Beta dose perturbation effects of typical stent types are almost negligible and can be corrected by an increased source dwell time if necessary. Guide wires produce effects which are clinically much more important and should therefore be retracted from the irradiation area.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

The cystic fibrosis transmembrane conductance regulator (CFTR) and the sulphonylurea receptor subunit (SUR) of the KATP channel are both members of the ATP-binding cassette (ABC) protein superfamily. Many compounds that open or block the KATP channel by binding to SUR also inhibit the CFTR Cl- current (ICFTR); an example in point is the chromanol-type KATP channel opener, cromakalim. The structurally related chromanol 293B (trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chromane), a blocker of the slow component of the delayed rectifier K+ current (IKs) in the heart, is also a weak inhibitor of KATP. This suggests that 293B may affect also ICFTR- We have addressed this question with human CFTR expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, 293B inhibited ICFTR with an IC50-value of 19 microM and Hill coefficient of 1.0; the inhibition was weakened by increasing concentrations of isobutyl-methylxanthine (IBMX). Patch-clamp recordings gave an IC50-value of 30 microM but showed a unusual variability in the sensitivity to 293B. The data show that 293B inhibits ICFTR and suggest that the mechanism of inhibition may depend on the phosphorylation state of the CFTR protein. The concentrations required for inhibition of ICFTR are three- to fivefold higher than those reported for inhibition of KvLQT1 + minK expressed in Xenopus oocytes. Since CFTR is expressed also in cardiac myocytes, the effects of 293B in these cells must be analysed with caution.

The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K+ channel (KATP channel), [3H]-PKF217 - 744 (3S,4R)-N-[3,4-dihydro-2,2-dimethyl-3-hydroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-3H]pyridinecarboxamide with a specific activity of 50 Ci mmol(-1) is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively. PKF217 - 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]-P1075, to SUR2B (Ki=26 nM). The kinetics of [3H]-PKF217 - 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9x10(6) M(-1) min(-1) and 0.09 min(-1), respectively. Binding of [3H]-PKF217 - 744 to SUR2B/2A was activated by MgATP (EC50 approximately 3 microM) and inhibited (SUR2B) or enhanced (SUR2A) by MGADP: Binding of [3H]-PKF217 - 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]-P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B-[3H]-PKF217 - 744 complex. The data show that the affinity of [3H]-PKF217 - 744 binding to SUR2B is approximately 6 times lower than that of [3H]-P1075. This is due to a surprisingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.

The effects of the fluoresceine derivative, phloxine B, on the Cl(-) current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. In whole oocytes, the CFTR Cl(-) current (I(CFTR)) was activated by superfusion with isobutylmethylxanthine and forskolin. I(CFTR) was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited I(CFTR) weakly. In excised inside-out macropatches, I(CFTR) was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01 - 3 microM), applied after activation, increased I(CFTR) within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC(50) value of approximately 1 microM. In the absence of cPKA, phloxine B did not stimulate I(CFTR). In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). The data show that phloxine B modulates I(CFTR) by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell.

An experiment was carried out to reevaluate the response of LiF TLD-100 rods (1 mmx1 mmx6 mm) at different depths in a water substituting phantom to provide an answer to a prevailing controversy about the over-response of LiF to the softened photon spectra of 192Ir HDR source at depths in phantom due to its photon energy dependence. Claims of some authors that LiF TLDs over-responds by 8.5% at 10 cm depth in phantom, necessitating depth-dependent correction factors even for an 192Ir source and of some others for no over-response were evaluated. The over-response of LiF TLD-100 rods, against a calibrated ion chamber having a photon energy-independent response within 2%, was found to be not exceeding 2.5% at a depth of 10 cm in the phantom as compared to a depth at 1 cm, for a precision of the order of +/- 1% (1sigma) in the TLD measurements. By using ISO equivalent photon beams, photon energy dependence of the dosimeters was evaluated and for LiF TLD-100 rods it was found to be in close agreement (within 3%) with the ratios of mass energy absorption coefficients of LiF and water in the range of effective photon energy from 26 keV to 1.25 MeV. Parameters that could contribute to the discrepancy in the reported values of experimental results have been discussed.

The effects of minoxidil sulphate, an "atypical" K(ATP) channel opener, and bimakalim, a benzopyran-type classical K(ATP) channel opener, on guinea pig airways in vitro and in vivo and on isolated portal veins from rats and guinea pigs were compared. Minoxidil sulphate inhibited the spontaneous activity of isolated guinea pig and rat portal vein preparations with pD2 values of 7.83+/-0.08 and 7.14+/-0.03, respectively (Emax=100% in both preparations). Bimakalim caused a more potent inhibition with pD2 values of 8.80+/-0.05 and 8.20+/-0.04, respectively (Emax=100% in both preparations). Minoxidil sulphate reduced the spontaneous tone of isolated guinea pig tracheal rings with a pIC50 value of 3.92+/-0.02 and the same efficacy as isoprenaline. Bimakalim was more potent (pIC50=7.25+/-0.02) but less efficacious (Emax=75% of the Emax of isoprenaline). The airway relaxant effect of bimakalim, but not minoxidil sulphate, was antagonised by glibenclamide (pA2=7.50) at concentrations above 0.1 microM. Bombesin-induced bronchoconstriction in anaesthetised, ventilated, normoreactive guinea pigs (measured as increase in total lung resistance) was dose-dependently reversed by intratracheally (i.t.) administered bimakalim (ED50=4 microg/kg; Emax=92% of maximally possible inhibition), but not by minoxidil sulphate, at doses up to 1 mg/kg i.t. In the same animals, following i.t. administration of higher doses, both minoxidil sulphate and bimakalim reduced blood pressure. Airways hyperreactivity to histamine induced by acute treatment of guinea pigs with immune complex was dose-dependently reversed by bimakalim (ED50=0.5 microg/kg i.t., Emax=100%). This effect was antagonised by glibenclamide (30 mg/kg i.v.). Minoxidil sulphate had a biphasic effect on airways hyperreactivity: at 1 microg/kg i.t., airways hyperreactivity was augmented, whereas at doses above 3.2 microg/kg i.t. it caused reversal of airways hyperreactivity. Both of the effects of minoxidil sulphate were insensitive to glibenclamide (30 mg/kg i.v.). It is concluded that the pharmacological profile of minoxidil sulphate in guinea pig airways is completely different from that of classical K(ATP) channel openers such as bimakalim. Minoxidil sulphate is either only weakly active or even inactive at K(ATP) channels in guinea pig airways or interacts with these channels in a different manner. The current results are consistent with there being differences between the K(ATP) channels in airways and blood vessels.

A fast dosimetry system based on plastic scintillator detectors has been developed which allows three-dimensional measurement of the radiation field in water of beta-sources appropriate for application in cardiovascular brachytherapy. This system fulfills the AAPM Task Group 60 recommendations for dosimetry of cardiovascular brachytherapy sources. To demonstrate the use of the system, measurements have been performed with an 90Y-wire source. The dose distribution was determined with a spatial resolution of better than 0.2 mm, with only a few minutes needed per scan. The scintillator dosemeter was absolutely calibrated in terms of absorbed dose to water with a precision of +/-7.5%. The relative precision achievable is +/-2.5%. The response of the system is linear within +/-2% for dose rates from 0.5 mGy s(-1) to 500 mGy s(-1).

ATP-sensitive K(+) channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K(+) channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K(+) channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37 degrees C. The equilibrium dissociation constants (K(D)) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K(D) value of the opener P1075 (N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine) ( approximately 5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC(50) approximately 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K(+) channel in vascular tissue; and 4) the K(D) value of GBC binding to the octameric channel is 7 times lower than the IC(50) value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

A new approach to optimize curative eye plaque brachytherapy is presented. The application of ophthalmic plaques is a common therapy modality for small and medium sized intraocular tumors. At Essen University Hospital eye applicators with photon emitting 125I seeds are used for the treatment of tumors with a thickness from 5 to 10 mm. Our clinical experiences indicate that the dose distributions of these applicators-used so far worldwide-are not optimal. A steeper dose falloff would meet the radiobiological requirements better, to provide the eradication of all tumor cells as well as sufficient occlusion of tumor supplying blood vessels. Our investigations for eye plaque optimization are based both on measurements and Monte Carlo simulation. For fast dosimetric measurements we have built a computer controlled device which allows reading out, directly and simultaneously, 16 1 mm3 scintillators. For the numerical simulations of the dose distribution of 125I eye plaques we have adapted a Monte Carlo program originally developed to calculate the synchrotron radiation in particle physics. We have investigated the influence of geometrical as well as physical eye plaque parameters on the dose distribution: Shielding of the primary radiation, penumbra modification, and energy conversion by exploiting fluorescence x-radiation have been considered. New types of fluorescence eye applicators have been designed which are more suitable for the prevention of radiopathic effects on structures at risk.

After phosphorylation by protein kinase A and in the presence of ATP, the cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel. In this study we have examined the effects of suramin on the CFTR Cl- current (I(CFTR)) in excised inside-out macropatches from Xenopus oocytes expressing human CFTR; glibenclamide, the standard inhibitor of I(CFTR), and some congeners were tested in comparison. I(CFTR) was activated by addition of the catalytic subunit of protein kinase A and MgATP to the bath. Suramin inhibited I(CFTR) with an IC50 value of 1 microM and a Hill coefficient close to 1; the inhibition showed little voltage dependence and was easily reversed upon washout of the drug. In comparison, glibenclamide inhibited I(CFTR) with an IC50 value of approximately 20 microM. When tested against I(CFTR) in whole oocytes, bath application of suramin was ineffective whereas glibenclamide was about four times weaker than in the inside-out patch configuration. The data show that suramin is the most potent inhibitor of CFTR yet described and suggest that the compound approaches its site of action from the cytosol.

1. Openers of the ATP-sensitive potassium channel (KATP channel) increase and blockers decrease renin secretion. Here we report the effects of levcromakalim (LCRK, a channel opener) and glibenclamide (GBC, a blocker) on membrane potential, whole-cell current and the cytoplasmic Ca2+ concentration of renin-secreting cells (RSC). Studies were performed on afferent arterioles from the kidney of Na+-depleted rats. 2. As monitored with the fluorescent oxonol dye DiBAC4(3), LCRK (0.3 and 1 microM) induced a hyperpolarization of approximately 15 mV which was abolished by GBC (1 microM). 3. Whole-cell current-clamp experiments showed that RSC had a membrane potential of -61 +/- 1 mV (n = 16). LCRK (1 microM) induced a hyperpolarization of 9.9 +/- 0.2 mV (n = 16) which, in the majority of cells, decreased slowly with time. 4. Capacitance measurements showed a strong electrical coupling of the cells in the preparation. 5. At -60 mV, LCRK induced a hyperpolarizing current in a concentration-dependent manner with an EC50 of 152 +/- 31 nM and a maximum current of about 200 pA. 6. Application of GBC (1 microM) produced no effect; however, when applied after LCRK (300 nM), GBC inhibited the opener-induced hyperpolarizing current with an IC50 of 103 +/- 36 nM. 7. LCRK (0.3 and 1 microM) did not significantly affect the cytoplasmic Ca2+ concentration either at rest or after stimulation by angiotensin II. 8. The data show that LCRK induces a GBC-sensitive hyperpolarizing current in rat RSC. This current presumably originates from the activation of KATP channels which pharmacologically resemble those in vascular smooth muscle cells. The stimulatory effect of KATP channel opening on renin secretion is not mediated by a decrease in intracellular Ca2+ concentration.

KATP channels are heteromeric complexes of inwardly rectifying K+ channel subunits and sulfonylurea receptors (SURs). SUR2A and SUR2B, which differ within the carboxyl terminal exon 38, are characteristic for the cardiac and smooth muscle type channels, respectively. Here we compare binding of the tritiated KATP channel opener, [3H]P1075, to membranes from human embryonic kidney (HEK) cells transfected with murine SUR2A and 2B at 37 degrees C. Binding to both SURs required addition of Mg2+ and ATP in the low micromolar range. In the presence of MgATP, micromolar concentrations of MgADP, formed by the ATPase activity of the membrane preparation, increased binding to SUR2A but inhibited binding to SUR2B. Decreasing temperatures strongly reduced [3H]P1075 binding to SUR2A, whereas binding to SUR2B was increased in a bell-shaped manner. Kinetic experiments revealed a faster dissociation of the [3H]P1075-SUR2A complex, whereas the association rate constants for [3H]P1075 binding to SUR2A and 2B were similar. Openers inhibited [3H]P1075 binding to SUR2A with potencies approximately 4 times lower than to SUR2B; in contrast, glibenclamide inhibited [3H]P1075 binding to SUR2A approximately 8 times more potently than to SUR2B. The data suggest that SUR2A and 2B represent the opener receptors of cardiac and vascular smooth muscle KATP channels, respectively, and show that MgADP is an important modulator of opener binding to SUR. The different carboxyl termini of SUR2A and 2B lead to differences in the MgADP dependence and the thermodynamics of [3H]P1075 binding, as well as in the affinities for openers and glibenclamide, underlining the importance of this part of the molecule for KATP channel modulator binding.

The risk of restenosis, main late effect limiting the success of percutaneous transluminal coronary artery angioplasty, can be reduced significantly by vascular radiotherapy, subsequent to PTCA. This discovery lead to the development of new irradiation techniques. Endovascular brachytherapy is the choice in treatment of coronary artery stenosis. Successful irradiation, however, requires precise treatment planning. This review addresses the physical possibilities and problems of intravascular brachytherapy planning, and the radiobiologically based definition of the target volume and of structures at risk. Recommendations for dose specification, recording and reporting are given. The criteria for selecting a vascular radiotherapy technique are discussed as well as the possibilities of dosimetric treatment planning and quality assurance based on precise plastic scintillator dosimetry and intravascular ultrasound. Radiation protection and safety must be reconsidered prior to the usage of therapeutic radiation sources in the catheter laboratory and for the decision about emergency plans. Finally, the design of clinical trials, the role of medical physicists, and the future of irradiation treatment of stenosis is discussed.

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N-1-adamantyl-N'-cyclohexylhyd rochloride) with the ATP-sensitive K+ channel (K(ATP) channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K(ATP) channels; this group is lacking in furosemide. U-37883A blocks several types of K(ATP) channels. The interaction with the vascular K(ATP) channel was probed in binding studies, 86Rb+ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K(ATP) channel inhibitor [3H]glibenclamide and of the opener [3H]P1075 with IC50 values of 19 and 45 microM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In 86Rb+ efflux experiments, the loop diuretics, at microM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated 86Rb+ efflux, a qualitative measure of K(ATP) channel opening, was inhibited by U-37883A and torasemide with IC50 values of 0.06 and 130 microM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC50 values between 6 and 10 microM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 microM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 microM. The data show that torasemide interferes specifically with the binding of the K(ATP) channel modulators [3H]glibenclamide and [3H]P1075 and with the K(ATP) channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K(ATP) channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced 86Rb+ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs.

1. The binding of [3H]-P1075, a potent opener of adenosine-5'-triphosphate-(ATP)-sensitive K+ channels, was studied in a crude heart membrane preparation of the rat, at 37 degrees C. 2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [3H]-P1075 binding with an EC50 value of 100 microM and a Hill coefficient of 1.4. 3. In saturation experiments [3H]-P1075 binding was homogeneous with a KD value of 6+/-1 nM and a binding capacity (Bmax) of 33+/-3 fmol mg(-1) protein. 4. Upon addition of an excess of unlabelled P1075, the [3H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13+/-0.01 min(-1). If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030+/-0.003 nM(-1) min(-1). From the kinetic experiments the KD value was calculated as 4.7+/-0.6 nM. 5. Openers of the ATP-sensitive K+ channel belonging to different structural classes inhibited specific [3H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta. 6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [3H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC50 value of glibenclamide being approximately 90 nM; affinities for the low affinity component were in the microM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC50 value of 6 microM, a maximum inhibition of 94% and a Hill coefficient of 1.5. 7. It is concluded that binding studies with [3H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [3H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR2A) and vascular smooth muscle cells (SUR2B), differs from that expected for SUR2A and SUR2B.

The interaction between the cytoskeleton and the ATP-sensitive K+ channel (KATP channel) was studied in rat aortic rings by examining the binding of the sulphonylurea blocker, 3H-glibenclamide, and of the opener, 3H-P1075. The actin cytoskeleton disrupting agents, cytochalasin D (1 microM) and latrunculin B (1 microM), abolished the high affinity component of 3H-glibenclamide binding. Preincubation with the actin cytoskeleton stabilizing agent, phalloidin (10 microM) prevented the effect of cytochalasin D. In contrast, binding of the opener, 3H-P1075, and inhibition of this binding by glibenclamide, were unaffected by cytochalasin D (3 microM). Colchicine (100 microM), which disassembles microtubules, had no effect on the binding of 3H-glibenclamide and 3H-P1075. The data show that high affinity binding of glibenclamide, which mediates the effects of the sulphonylurea in this preparation, requires the presence of an intact actin cytoskeleton. Binding of the opener is unaffected by the state of the cytoskeleton and preserves a conformational state in which high affinity binding of glibenclamide to the sulphonylurea receptor can occur.

Despite numerous efforts in catheter technology and procedural approaches the problem of restenosis in interventional cardiology persists. Although the implantation of coronary stents has significantly reduced restenosis rates based on the inhibition of elastic recoil, intimal proliferation as the second major mechanism for postinterventional restenosis could not effectively be suppressed. Intimal proliferation is the response to vessel injury following interventional procedure, e.g. balloon angioplasty. It results in the adhesion of mono- and lymphocytes which themselves trigger the colonisation of myofibroblasts. Intracoronary irradiation seeks to prevent this proliferative process as it destroys or irreversibly alters DNA structures of cells at the site of balloon injury. The antiproliferative effect depends on the irradiation dosis, the timing and the cell cycle phase. Mainly beta- and gamma-radiation is used for intracoronary irradiation. Beta-emitters are characterized by a sharp decline of dose rate within millimeters from the actual source. The exposure to surrounding tissue as well the catheter staff can be kept to a minimum. The high intensity of beta-emitters allow a short treatment period of minutes to gain an effective radiation dose to the target. In contrast, gamma-emitters have a low radial dose distribution resulting in high dosage even centimeters away from the source. These emitters require additional shielding in the catheter laboratory and lead to excessive whole body doses. To achieve a sufficient dose in the target tissue, irradiation times of more than 20 minutes are necessary which prolongs the interventional procedure substantially. At present, catheter based systems or radioactive implantable stents are available to deliver the required dose. Catheter based systems seem more flexible in a number of considerations. On the other hand they require a substantial amount of hardware. Beta-emitting stents are implanted via a conventional stent delivery system with small shielding modifications. However, stents emit an inhomogeneous radiation profile due to the mesh-like structure. In addition, not every lesion can be reached by a stent nor does every lesion require a stent solely to deliver radiation. External irradiation is presently not recommended due to its ineffectiveness and the high rate of side effects. In the experimental setting the porcine model comes closest to the clinical situation in man. Animal experiments have demonstrated the effective reduction of intimal proliferation using beta- and gamma-sources in a wide dose range of 3 to 56 Gy. Although the initial and early results are convincing little is know about the long term results. Only few studies have been and are currently performed in patients. Some of these investigations demonstrate a significant reduction of restenosis rate after 6 months. Again, information on long-term results are lacking. It has to be considered that perivascular fibrosis, which may occur with a delay of 5 to 10 years depending on the dosage, could curtail the initial success. Intracoronary irradiation is a promising method for the prevention of restenosis. The dose finding with respect to the dose effect relation, the determination of the therapeutic window and the timing of irradiation have to be further defined in the clinical setting. Nevertheless, intracoronary irradiation remains high on the priority list in fighting restenosis.

1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.

In vascular smooth muscle, openers of ATP-dependent potassium channels (K(ATP) channels), such as P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the 86Rb+ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in 86Rb+ efflux induced by P1075 was taken as a qualitative measure of K+ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100 microM), abolished specific binding of [3H]-P1075 with an IC50 value of 7.6+/-1.2 microM; at 30 microM, the half time for inhibition was 38 min. Two other thioloxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2'-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1 mM), reduced [3H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In 86Rb+ efflux experiments, thimerosal (3 to 100 microM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC50 value of 7+/-1 microM. The inhibitory effect occurred with a half-time of approximately 8 min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation. The data show that oxidation of thiol groups interferes with the binding of the K(ATP) channel opener, P1075; concomitantly, the 86Rb+ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K(ATP) channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated 86Rb+ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity.

In rat aortic rings, P1075, an opener of ATP-dependent potassium channels (K(ATP) channels), produces relaxation and 86Rb+ efflux from preloaded tissues; the increase in 86Rb+ efflux qualitatively reflects K(ATP) channel opening. In this study we have investigated the effects of protein kinase C modulation on the 86Rb+ efflux stimulating, the vasorelaxant and the binding properties of P1075. Phorbol 12,13-dibutyrate (PDBu), a direct activator of protein kinase C, inhibited the 86Rb+ efflux produced by P1075 with an IC50 value of 20+/-2 nM. Phorbol 12-myristate 13-acetate (PMA), another stimulator of protein kinase C, was 150 times weaker in this respect whereas 4alpha-PDBu, the inactive stereoisomer of PDBu, was ineffective. Staurosporine (300 nM), an inhibitor of protein kinase C, induced a small but significant increase of P1075-induced tracer efflux and partially reversed the inhibitory effect of PDBu on P1075-stimulated tracer efflux. The vasorelaxant effect of P1075 was inhibited only to a moderate degree by PDBu at concentrations which inhibited P1075-induced 86Rb+ efflux to >90%; however, in the presence of PDBu, the relaxation kinetics of P1075 were increasingly slowed. The vasorelaxant effect of P1075 in the presence of PDBu was still sensitive to inhibition by glibenclamide (100 nM), the standard inhibitor of the K(ATP) channel openers. Specific binding of [3H]-P1075 to rat aortic rings was unaffected by PDBu and PMA even in the micromolar concentration range. The data show that stimulation of protein kinase C inhibits the K+ channel opening effect of P1075 in rat aorta and suggest that protein kinase C may exert a weak tonic inhibition on the K(ATP) channels in this vessel under quasiphysiological conditions. At concentrations of PDBu which essentially abolished P1075-induced tracer efflux, the glibenclamide-sensitive vasorelaxant effect of P1075 was slowed down but not prevented; this supports earlier suggestions that K+ channel openers are also able to relax smooth muscle cells by a mechanism independent of K(ATP) channel opening.

Diabetes mellitus is associated with major vascular complications. It was the aim of this study to examine the function of the ATP-sensitive K+ channel (K(ATP) channel) in aortic rings prepared from diabetic rats and from age-matched controls. Diabetes was induced by injection of streptozotocin (60 mg/kg i.p.) and the animals were sacrificed 10 weeks after treatment. The binding of the K(ATP) channel opener, P1075 (N-cyano-N'-(1,1dimethylpropyl)-N"-3-pyridylguanidine), as well as the vasorelaxant and 86Rb+ efflux stimulating effects of the drug were measured. In endothelium-denuded rings from diabetic rats, the maximum contraction and sensitivity to noradrenaline were increased; in rings with intact endothelium, the acetylcholine-induced (endothelium-dependent) relaxation was similar in the two groups. In rings from diabetic rats the relaxation-concentration curve of the K(ATP) channel opener P1075 against noradrenaline was shifted rightwards by a factor of 1.3 and the maximum relaxation was reduced from 81 to 71% of initial tension (P <0.01). However, specific binding of 3H-P1075 was increased by 20% without a change in affinity, indicating that the number of binding sites for the opener was increased as a consequence of diabetes. In addition, P1075-induced 86Rb+ efflux, a qualitative measure of K(ATP) channel opening, was augmented by 50%. The data show that in the aorta from diabetic rats the K+ channel opening response to P1075 is markedly increased; however, the vasorelaxant effect to the K(ATP) channel opener is slightly impaired. A possible explanation of these findings is that the vasorelaxant mechanisms (which are in part independent of plasmalemmal K(ATP) channel opening) may be altered; alternatively, the link between membrane potential and smooth muscle tone may be changed in this model of insulin-dependent diabetes mellitus.

The effect of activators of protein kinase A on membrane K+ permeability and the interaction of these compounds with cromakalim, an opener of ATP-sensitive K+ channels (K(ATP) channels), were investigated. Membrane K+ permeability was assessed by measuring 86Rb+ efflux from rings of rat aorta. Forskolin, an activator of adenylate cyclase, and isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, induced small, concentration-dependent increases in tracer efflux up to 20-40% over the basal level. The effect of forskolin was abolished by the K+ channel blocker tedisamil (1 microM) and partially inhibited by glibenclamide (1 microM), a relatively selective blocker of K(ATP) channels. Further studies were conducted in the presence of 35 mM KCI in the bath in order to increase the size of the 86Rb+ efflux stimulated by forskolin and IBMX. At high concentrations, these compounds produced a biphasic effect with a peak increase being followed by a lower plateau value. Glibenclamide inhibited the 86Rb+ efflux response to forskolin and IBMX by 50-80%. The K+ channel blockers tedisamil (1 microM), Ba2+ (1 mM) and tetraethylammonium (10 mM) also reduced the peak response to forskolin by about 50% and abolished or greatly inhibited the plateau response. In addition to the small effect on basal 86Rb+ efflux, forskolin (0.3 microM) increased cromakalim-induced 86Rb+ efflux 3.4 times. At higher concentrations, however, a concentration-dependent inhibition was observed with an IC50 value of 7.6 +/- 0.4 microM. 1,9-dideoxyforskolin, which does not increase cAMP, increased neither basal nor cromakalim-induced 86Rb+ efflux; however, it inhibited cromakalim-stimulated tracer efflux with an IC50 value of 22 +/- 2 microM. It is concluded that forskolin and IBMX, probably by increasing intracellular cAMP levels, induce a 86Rb+ efflux from rat aorta, the major part of which is glibenclamide-sensitive and may pass through K(ATP) channels. In addition, low concentrations of forskolin greatly facilitate the K(ATP) channel opening effect of cromakalim whereas high concentrations block the channel; this blocking effect of forskolin is unrelated to the cAMP elevating action.

1. The binding of the sulphonylurea [3H]-glibenclamide, a blocker of adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP channels), was studied in endothelium-denuded rings from rat aorta. 2. [3H]-glibenclamide labelled two classes of binding sites with KD values of 20 +/- 5 nM and 32 +/- 1 microM. The high affinity component, which comprised 17% of total binding at 1 nM [3H]-glibenclamide, had an estimated binding capacity of 150 fmol mg-1 wet weight. 3. Other sulphonylureas such as glipizide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited high affinity [3H]-glibenclamide binding with the potencies expected from their K+ channel activity. At very high concentrations, AZ-DF 265 and glipizide started to interact also with the low affinity component of [3H]-glibenclamide binding. 4. Openers of the ATP-sensitive K+ channel belonging to different structural groups inhibited only the high affinity [3H]-glibenclamide binding; the potencies in this assay were similar to those obtained in functional (i.e. vasorelaxation) studies. 5. High affinity [3H]-glibenclamide binding was abolished by prolonged hypoxia combined with metabolic inhibition. 6. The data indicate that the high affinity component of [3H]-glibenclamide binding mediates the block of the KATP channel by the sulphonylureas in rat aorta; hence, it represents the sulphonylurea receptor in this vessel. The pharmacological properties of this binding site resemble those of the binding site for the openers of the KATP channel; present evidence suggests that these two classes of sites are negatively allosterically coupled.

The kidney is endowed with ATP-sensitive K+ channels (KATP channels) both at the vascular and at the epithelial level. In this study we have characterized the binding of the sulphonylurea glibenclamide, the most widely used blocker of KATP channels, in rat isolated glomeruli. In metabolically intact glomeruli, 3H-glibenclamide labelled two different binding components with affinities of 47 +/- 12 nM and 10 +/- 1 microM and estimated binding capacities of 1.2 +/- 0.1 and 501 +/- 11 pmol/mg protein, respectively. 3H-glibenclamide binding was inhibited differentially by other sulphonylureas (tolbutamide, glibornuride, gliquidone and glipizide) and benzoic acid analogues such as meglitinide, AZ-DF 265 and UL-DF 9. Sulphonylureas interacted with the high affinity component and, in some cases, also with the low affinity component whereas the benzoic acid derivatives inhibited exclusively low affinity glibenclamide binding. Severe metabolic stress affected both components of glibenclamide binding by shifting high affinity binding to the right and reducing the capacity of the low affinity component. Disruption of the cytoskeletal actin filaments by cytochalasin B and D mimicked the effect of metabolic stress on the high affinity component but left the low affinity component unchanged. In crude membranes, the affinity of the first component was again reduced and a major loss of the low affinity sites was observed. The data show that the two binding components of glibenclamide binding in rat isolated glomeruli have very different properties. The high affinity component is not recognized by the benzoic acid derivatives; its affinity is modulated by cell metabolism and the actin component of the cytoskeleton. The low affinity sites are, in their majority, cytosolic. The function and cellular localization of the high affinity sites are under further study.

Oral vaccination against poliomyelitis, which was carried out worldwide, lead to eradication of poliomyelitis in the United States, in South America and parts of Europe; in other parts of the world, paralytic poliomyelitis is still a severe risk of health. In those countries where poliomyelitis has been eradicated, it is presently discussed whether the vaccination schedules should be changed to an inactivated polio vaccine (IPV), as in polio-free countries only cases of paralytic poliomyelitis after vaccinations have been reported. Behringwerke's data from a 30-year period of analysing adverse drug reaction reveal the following: using the trivalent oral polio vaccine (OPV), based on WHO case definition, the risk for vaccine-associated paralytic poliomyelitis with permanent damage is approximately 1 case for 4.5 million vaccinations (0.22 per million) in vaccinees, and approximately 1 case for 11 million (0.09 per million) in contact persons. This low risk is in line with the data ascertained worldwide.

ATP-sensitive K+ channels (KATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [3H]-P1075 ([3H]-N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine), a selective opener of KATP channels, in rat glomerular preparations. Equilibrium (saturation, competition) and kinetic experiments indicated that [3H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg-1 glomerular protein. The association rate constant of the complex was 6,5 x 10(7) M-1 min-1; dissociation occurred with a half-time of 6.2 min. Specific [3H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [3H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [3H]-P1075 binding was inhibited by representatives of the different classes of KATP channel openers and by sulphonylurea-type blockers with inhibition constants similar to those obtained in rat aortic rings. It is concluded that rat glomerular preparations possess specific binding sites for KATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 microns-2) is much higher than that of KATP channels in smooth muscle.

ATP-sensitive K+ channels (KATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K+ which, in turn, is a major determinant of cell membrane potential. KATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic beta-cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell. In the renal tubular system, KATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K+ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the KATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed. The pharmacology of KATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of KATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the KATP channels in the pancreatic beta-cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K+ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the KATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.

The K+ channel openers activate ATP-sensitive K+ channels (KATP) in vascular smooth muscle and induce relaxation. In this study, the relationship between these two effects was examined in rings of rat aorta using levcromakalim and minoxidil sulfate as the openers and Ba2+ as the K+ channel blocker; K+ channel opening was assessed by determining the rate constant of 86Rb+ efflux from the preparation. Ba2+ inhibited the 86Rb+ efflux stimulated by levcromakalim in a noncompetitive manner with an IC50 value of 29 microM and a Hill-coefficient of 1.2. At concentrations >300 microM, Ba2+ increased the tension of rat aortic rings concentration-dependently. Levcromakalim relaxed contractions to Ba2+ (0.5 and 1 mM) with potencies similar to those determined against KCl (25 mM) or noradrenaline as spasmogens (EC50 values 15-40 nM). The vasorelaxant effect against Ba2+ was inhibited by the KATP channel blockers, glibenclamide and tedisamil, and abolished in depolarizing medium (55 mM KCl). At 3 mM Ba2+, levcromakalim was still able to transiently induce complete relaxation; however, within 1 h oscillations in tension developed, leading to a stable level of only 15% relaxation. A similar level of relaxation was achieved against 10 mM Ba2+ whereas the combination of 0.5 mM Ba2+ and 3 microM tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the KATP channel opener the results of the 86Rb+ efflux and tension experiments were similar to those obtained with levcromakalim. It is concluded that Ba2+ is more potent in inhibiting the K+ channel opening than the vasorelaxant effects of the openers. On the basis of the 86Rb+ efflux experiments it is estimated that at least 97% of the channels opened by the activators can be blocked without major effects on vasorelaxation suggesting a dissociation between the two effects. However, if the block is pushed to extremes (> or = 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.

1. The effects of analogues and stimulators of cyclic AMP on the 86Rb+ efflux-stimulating and binding properties of P1075, an opener of ATP-dependent potassium channels, were studied in rat aortic rings. The increase in 86Rb+ efflux stimulated by P1075 was taken as a qualitative measure of K+ channel opening. 2. Forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and dibutyryl-cyclic AMP (db-cyclic AMP), a membrane permeant cyclic AMP-analogue, relaxed rat aortic rings contracted by noradrenaline with EC50 values of 0.06, 2 and 10 microM, respectively. 3. Forskolin, IBMX and db-cyclic AMP produced concentration-dependent increases of the 86Rb+ efflux induced by P1075 (50 nM) by up to twofold with EC50 values of about 0.1, 1.7 and 81 microM. At these concentrations the agents had little effect on the basal rate of 86Rb+ efflux. 4. The 86Rb+ efflux produced by P1075 in the presence of the cyclic AMP stimulators was inhibited by glibenclamide, a blocker of ATP-sensitive potassium channels. 5. IBMX (100 microM) induced a leftward shift of the concentration-86Rb+ efflux curve of P1075 without increasing the maximum. The enhancements of P1075-stimulated 86Rb+ efflux produced by combinations of forskolin and IBMX were either additive or less than additive. 6. The protein kinase A inhibitor, H-89, inhibited P1075-stimulated 86Rb+ efflux in the presence of IBMX significantly more than in the absence of IBMX, suggesting that the effect of increased cyclic AMP levels is mediated by protein kinase A. 7. At high concentrations, forskolin and IBMX slightly increased basal 86Rb+ efflux and inhibited the tracer efflux induced by P1075.8. Binding of [3H]-P1075 to rat aortic rings was either unaffected or inhibited by forskolin, IBMX and db-cyclic AMP.9. This study shows that moderate stimulation of the cyclic AMP system potentiates the K+ channel opening effect of P1075 by activation of protein kinase A. The fact that binding of [3H]-P1075 remains unchanged or is diminished favours the hypothesis that the K'channel openers activate ATP-dependent K+ channels by an indirect mechanism.

A simple and fast, but sensitive TLD method for the measurement of energy and homogeneity of therapeutically used electron beams has been developed and tested. This method is based on the fact that when small thicknesses of high-Z absorbers such as lead are interposed in the high-energy electron beams, the transmitted radiation increases with the energy of the electron beams. Consequently, the ratio of readouts of TLDS held on the two sides of a lead plate varied sharply (by factor of 70) with a change in energy of the electron beam from 5 MeV to 18 MeV, offering a very sensitive method for the measurement of the energy of electron beams. By using the ratio of TL readouts of two types of TLD ribbon with widely different sensitivities, LiF TLD-700 ribbons on the upstream side and highly sensitive CaF2:Dy TLD-200 ribbons on the downstream side, an electron energy discrimination of better than +/- 0.1 MeV could be achieved. The homogeneity of the electron beam energy and the absorbed dose was measured by using a jig in which the TLDS were held in the desired array on both sides of a 4 mm thick lead plate. The method takes minimal beam time and makes it possible to carry out measurements for the audit of the quality of electron beams as well as for intercomparison of beams by mail.

Potassium channel opening is a physiological mechanism which excitable cells exploit to maintain or restore their resting state. Thus drugs that open vascular potassium channels have the potential to restrain or prevent contractile responses to excitatory stimuli or clamp the vessel in a relaxed condition. Hence, potassium channel openers, such as aprikalim and levcromakalim, relax agonist precontracted vascular preparations in vitro and lower systemic and regional vascular resistances in intact animals. Glibenclamide, a blocker of ATP sensitive potassium (KATP) channels, antagonises these effects. The main vasorelaxant mechanism of the potassium channel openers is to increase the potassium efflux through opening plasmalemmal potassium channels, which repolarises and/or hyperpolarises the membrane. This effect lowers the opening probability of voltage dependent calcium channels, restrains agonist induced calcium release from intracellular sources through inhibition of inositol trisphosphate formation, decreases the sensitivity of intracellular contractile elements to calcium, and accelerates the clearance of intracellular calcium via the Na+/Ca+ exchanger. Experimental evidence indicates that mechanisms not linked to potassium channel opening may also contribute to the potassium channel opener induced vasorelaxation; these remain to be clearly defined (for example, inhibition of the refilling of intracellular calcium stores). Potassium channel openers displace the binding of 3H-P1075, a potent potassium channel opener, in myocytes and intact rings from the rat aorta. In patches from vascular myocytes, potassium channel openers primarily open a small conductance (10-20 pS) KATP channel which is gated by [ATP]i and particularly by nucleotide diphosphates and magnesium.(ABSTRACT TRUNCATED AT 250 WORDS)

During the past decade, a group of chemically heterogeneous compounds known as the K+ channel openers has emerged. These compounds open a certain class of K+ channels (ATP-sensitive K+ channels) in the sarcolemma of vascular smooth muscle cells, which leads to hyperpolarization of the cell membrane and relaxation of the tissue. The mechanisms by which hyperpolarization affects smooth muscle contraction and contractility can thus be examined. Hyperpolarization induced by these K+ channel openers prevents Ca2+ entry through voltage-operated Ca2+ channels. Surprisingly, and by mechanisms not yet defined, hyperpolarization of the cell also reduces agonist-induced accumulation of inositol 1,4,5-trisphosphate (and consequently, Ca2+ mobilization from intracellular stores), and the Ca2+ sensitivity of the contractile apparatus. In addition, recent evidence reviewed here by Ulrich Quast suggests that the K+ channel openers possess further mechanisms of vasorelaxation not linked to the opening of plasmalemmal K+ channels.