Biomedical subjects

M Schramm

Publications and source records attributed to M Schramm.

At least 19 recordsLinked to original sources

PubMed2003

BAY 41-2272: a stimulator of soluble guanylyl cyclase induces nitric oxide-dependent penile erection in vivo.

OBJECTIVES: To determine the effectiveness of BAY 41-2272 on penile erections in an in vivo rabbit model. The nitric oxide (NO)-dependent increase of intracellular cyclic guanosine monophosphate (cGMP) by cGMP-phosphodiesterase (PDE5) inhibition has been shown to be an effective mechanism in the treatment of erectile dysfunction. Direct, NO-independent stimulation of soluble guanylyl cyclase should also lead to elevated cGMP levels in tissues and could be an attractive alternative therapeutic option for the treatment of erectile dysfunction. BAY 41-2272 is a novel non-NO-based direct stimulator of soluble guanylyl cyclase that activates purified enzyme in a synergistic fashion with NO. METHODS: BAY 41-2272 was administered to conscious rabbits intravenously (IV) and orally (PO). Erection was assessed in a time-dependent manner by measuring the length of the uncovered penile mucosa. Erections were evaluated in the absence and presence of NO (with intravenous sodium nitroprusside [SNP] as the NO donor). RESULTS: BAY 41-2272 only induced weak penile erections in conscious rabbits after IV (1 mg/kg) and PO (10 mg/kg) administration in the absence of an NO donor. However, the efficacy of BAY 41-2272 was potentiated by the simultaneous administration of SNP. Through simultaneous SNP administration, the effective doses of BAY 41-2272 were reduced significantly (minimal effective dose 0.1 mg/kg IV and 1 mg/kg PO). CONCLUSIONS: The results of this study clearly demonstrated the effect of BAY 41-2272 on penile erection in the conscious rabbit model after PO and IV administration. The time-course and onset of erection was concurrent with the stimulation by exogenous NO (SNP), suggesting that this new pharmacologic mechanism of soluble guanylyl cyclase stimulation could be used in the treatment of erectile dysfunction. Because the effect is increased by SNP, it can be expected that BAY 41-2272 would have enhanced activity during sexual arousal, when NO is produced endogenously.

Administration, Oral↗

PubMed2002

[The MACS-HMA hollow screw. An alternative possibility for stable implant anchoring in the vertebral body also for long fusions].

Our biomechanical in vitro tests compared the stability of the MACS HMA (Aesculap, Tuttlingen) implants to three established systems. The MACS HMA is a modular system consisting of porous hollow titanium screws with an outer diameter of 12 mm for monocortical use. We report the preliminary results of MACS HMA used for correction of scoliosis. All other implant systems used are designed with bicortical screws; one is for application with two screws/two rods and one uses a pullout-resistant nut behind the opposite cortex to increase stability. Significantly increased motion in the craniocaudal direction was recognized for bicortical standard screws (20 +/- 17 microns) compared to MACS HMA (10 +/- 11 microns) or the dual screw system (12 +/- 6 microns). Two-tailed t-test showed significantly higher stability for the dual screw system (4.2 kN) and the system with pullout-resistant nut (4.0 kN) compared to all other systems (p < 0.025 or higher). Bicortical implants (2.1-3.2 kN) and MACS HMA (2.6 kN) did not reveal significant differences in pullout strength. All biomechanical tests and in vivo use demonstrated favorable performance of MACS HMA implants.

Adolescent↗

PubMed2001

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272.

BACKGROUND: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase. RESULTS: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL. CONCLUSIONS: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Animals↗

PubMed2001

NO-independent regulatory site on soluble guanylate cyclase.

Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.

Amino Acid Sequence↗

PubMed2001

Pertussis toxin does not affect the time course of quantal release in crayfish and mouse muscle, but has other post- and presynaptic effects, especially on adenosine autoreceptors.

While G-proteins are involved in the synaptic release machinery and also can mediate inhibition of presynaptic Ca2+ channels, we find that pertussis toxin (PTX) does not affect the amount and the time course of quantal release from motor nerve terminals on crayfish or mouse muscle. Monoquantal excitatory currents (qEPSCs) were recorded that were elicited by constant depolarisation pulses to a terminal by means of a perfused macro-patch electrode. Although presynaptic effects of PTX on output and time course of release of quanta were absent, postsynaptically the rise time of qEPCs was increased and their decay time constant reduced. Adenosine (Ad) is known to inhibit quantal release in vertebrate motor nerve terminals via PTX sensitive G-proteins, and Ad is generated during nicotinic synaptic transmission by breakdown of the co-transmitter adenosine triphosphate (ATP). As reported by others, we found in mouse muscle inhibition of quantal release after application of Ad, but in addition late facilitation. Both these effects of Ad were blocked when the muscle was pre-incubated with PTX.

Adenosine Triphosphate↗

PubMed2001

Initial stability of modular acetabular components. Comparative in-vitro study with polyethylene and ceramic liners.

Modular acetabular components with alumina ceramic liners are currently used in total hip arthroplasty, but concerns have emerged regarding their high stiffness, which could cause impairment of stability, stress-shielding phenomena, and loosening. The purpose of the present biomechanical investigation was to compare the in-vitro initial stability of a modular press-fit acetabular component using a polyethylene liner and using an alumina liner. The initial stability was investigated by measuring the micromotion between the implant and the acetabulum during the application of physiological load (2.39 kN). The micromotion of the acetabular component was investigated in 10 acetabuli using a polyethylene liner and in 10 acetabuli using an alumina liner. Micromotion was assessed at the level of the Os ilium, Os pubis, and Os ischium using 3 electromagnetic transducers. The transducers have a sensitivity of 1 micron and a range of measurement of 500 microns. All implants have been fixed on human pelves made of polyurethane. Measurement of implant micromotion showed stable conditions at the level of the three main sectors of the acetabulum during all tests. No statistically significant differences of results were observed between the group of specimens with polyethylene liner and the group of specimens with alumina liner. The mean micromotion values of the uncemented cups were similar to the mean micromotion values of 10 cemented cups investigated to achieve comparative data of stability. In conclusion, the modular acetabular components inserted using an alumina liner showed a satisfactory initial stability in-vitro. The results do not contrast with those achieved using the same cup inserted with a polyethylene liner.

Acetabulum↗

PubMed2000

Patterns and determinants of blood lead during pregnancy.

The pattern of blood lead during pregnancy was investigated in a cohort of 195 women who, between October 1992 and February 1995, entered prenatal care at Magee-Womens Hospital in Pittsburgh, Pennsylvania, by week 13 of pregnancy. Blood was drawn as many as five times, once in each of the first two trimesters and a maximum of three times in the third trimester. Blood lead determinations were made by atomic absorption spectrophotometry. Potential sources or modifiers of lead exposure were collected by interviews, including sociodemographic, pregnancy history, occupational, and lifestyle data. Results confirmed a previously reported U-shaped curve in blood lead concentration during pregnancy as well as findings that blood lead levels increase with age, smoking, lower educational level, and African-American race and decrease with history of breastfeeding and higher intake of calcium. Additionally, interactions were found between time since last menstrual period and both maternal age and calcium. Specifically, older mothers showed steeper increases in blood lead concentrations during the latter half of pregnancy than did younger mothers, and intake of calcium had a protective effect only in the latter half of pregnancy, an effect that became stronger as pregnancy progressed. These findings provide further evidence that lead is mobilized from bone during the latter half of pregnancy and that calcium intake may prevent bone demineralization.

Adult↗

PubMed2000

Total hip arthroplasty using an uncemented femoral component with taper design: outcome at 10-year follow-up.

The aim of this study was to assess the clinical and radiological outcome of the cementless-system (CLS) femoral component designed by Spotorno with a taper design. Ninety-four patients (107 hips) were operated on from January 1987 to December 1988. The female/male ratio was 2/1, the mean age was 51 years (range 20-77 years). Clinical follow-up was obtained in 89 hips (83%). Five patients (7 hips, 6.5%) could not be traced. Nine patients (11 hips, 10%) had died before the minimum time of follow-up required for this study (10 years). All these hips still had their stem in place at the time of the last intermediate inquiry. The mean follow-up was 10.3 +/- 0.3 years. No stem required revision. According to the Harris score, 84% of hips were rated good or excellent, 14% fair, and 2% poor. The average Harris hip score was 88 at the time of the last follow-up. Fifteen patients (17%) reported occasional thigh pain. All patients reported complete relief of pain within the first 6 months after the operation. Radiologically, 95% of stems showed stable fixation by bony ingrowth, 5% by fibrous ingrowth. Seven (9%) uncemented acetabular components showed progressive migration. Three of those cups had been replaced in the meantime. Fifty-one (65%) of the CLS stems induced either no change in femoral bone density or only patchy loss of bone density localized to zone 1 or 7 according to Gruen, while 27 (35%) of the hips showed some reduction of bone density in the proximal diaphysis of the femur (zone 2 or 6). Severe progressive osteolysis of the femoral cortex underneath the lesser trochanter was found in 4 hips (5%) with coexistent loosening of the cup. In all of these hips, the Mecron cup had been used. At a mean follow-up of 10 years, the results of the CLS femoral component are comparable with those of modern techniques of cementing in primary total hip arthroplasty and with the long-term outcomes of other uncemented stems with tapered design. The long-term fixation of the cup remains an unsolved problem.

Adult↗

PubMed2000

Relaxation of human ureteral smooth muscle in vitro by modulation of cyclic nucleotide-dependent pathways.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

Colforsin↗

PubMed2000

Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder.

1. Recent studies have provided support for the notion that the high affinity neurotrophin receptor tyrosine receptor kinase B (trk B) may be involved in the treatment of depression. 2. Using a quantitative RT-PCR approach trk B mRNA levels were determined in brain material from cerebellum, temporal cortex, and frontal cortex of control specimen and patients with major depressive disorder, schizophrenia and bipolar disorder (15 subjects each). 3. Interestingly, elevated trk B mRNA levels were found in cerebellum (3.6-fold) in patients with major depressive disorder, reaching statistical significance (p=0.03). 4. The major depressive disorder-on drugs group differed from controls (p=0.006) in the cerebellum. 5. Since only patients with major depressive disorder received antidepressants, elevated trk B mRNA levels are possibly related to drug treatment.

Adult↗

PubMed2000

Stress and pregnancy among African-American women.

Research on stress tends to support an adverse effect on pregnancy outcomes, and suggests that the impact of these stressors is modified by social class and/or race. This study explicitly examined social factors such as experiences of discrimination, either racial or sexual, and neighbourhood crime as predictors of stress. We also examined cortisol and stress as predictors of blood pressure. A subsample of 94 African-American pregnant women, aged 18-39 years, who were enrolled in a longitudinal study of pregnancy and exposure to lead in the environment were used in this analysis. The women were patients at an obstetrics clinic at Magee Women's Hospital in Pittsburgh, PA, USA. Younger age, higher income, lower education and experiences of discrimination, both racial and sexual, were related to greater perceived stress; however, life events were not related to perceived stress. Higher income and urinary cortisol adjusted for creatinine were related to systolic blood pressure after the 36th week. As a body of evidence suggests that stress can have deleterious effects in both pregnant and non-pregnant women, future research should examine these forms of discrimination, especially racial discrimination, as a possible reason for the disparity in adverse pregnancy outcomes between African-American and white women.

Adolescent↗

PubMed2000

The vasodilator-stimulated phosphoprotein (VASP): target of YC-1 and nitric oxide effects in human and rat platelets.

The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser(239) and Ser(157)) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser(157) phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser(239), the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.

Animals↗

PubMed2000

Immunodetection of alpha1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney.

The calcium channel alpha1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of alpha1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac alpha1E was determined by SDS-PAGE and immunoblotting (218 +/- 6 kD; n = 3). Compared to alpha1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of alpha1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of alpha1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of alpha1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of alpha1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca(2+)-dependent secretion of peptide hormones such as ANP and urodilatin.

Alternative Splicing↗

PubMed2000

The Morse taper junction in modular revision hip replacement--a biomechanical and retrieval analysis.

All biomaterials used for total joint surgery are subjected to wear mechanisms. Morse taper junctions of modular hip revision implants are predilection sites for both fretting and crevice corrosion, dissociation and breakage of the components. The aim of this study is to quantify wear and study metallurgical changes of Morse taper junctions of in-vitro and in-vivo loaded modular revision stems. Three modular revision stems (MRP-Titan, Peter Brehm GmbH, Germany) were loaded by a servohydraulic testing machine. The loads and conditions used exceeded by far the values required by ISO-standard 7206. The tests were performed with maximum axial loads of 3,500 N to 4,000 N over 10-12 x 10(6) cycles at 2 Hz. Additionally, the female part of the taper junctions were coated with blood and bone debris. The free length of the implant was set to 200 mm. One other MRP stem was investigated after retrieval following 5.5 years of in-vivo use. All contact surfaces of the modular elements were assessed by visual inspection, optical microscopy and scanning electron microscopy (SEM). The degree of plastic deformation of the male part of the morse taper junction was determined by contouroscopy. None of the morse taper junctions broke or failed mechanically. Corrosion and wear affected all tapers, especially at the medial side. The retrieved implant showed no cracks and the amount of debris measured only one third of that for the stems tested in-vitro. The present retrieval and laboratory investigations have proven, that the morse taper junctions of the MRP-titanium stem are stable and resistant to relevant wear mechanisms. The longevity of the junctions for clinical use is given. If an optimal taper design is selected, the advantages of modular femoral components in total hip revision arthroplasty will outweigh the possible risks.

Biomechanical Phenomena↗

PubMed1999

Prophylaxis of secondary osteoarthrosis with spherical osteotomy in residual acetabular dysplasia. Analysis of predictive factors of success.

The aim of this study was to evaluate whether or not spherical acetabular osteotomy prevents progression of osteoarthrosis in hip joints with residual dysplasia and which radiological parameters can be used postoperatively as predictive factors concerning the outcome. Sixty-eight out of 78 joints were re-investigated with a mean follow-up of 11.2 years after the index operation. Twenty-three Wagner type 1 and 45 type II osteotomies were performed. At surgery, the mean age of the patients was 26 years; 90% of joints showed no or mild degenerative changes, 26% presented with a good or excellent Harris hip score. At follow-up, 28% of patients had improved in function, mean Harris hip score rated 75 points. The mean center-edge (CE) angle improved from -4 degrees to 18 degrees, and the anterior center-edge (ACE) angle from -4 degrees to 26 degrees. The weight-bearing zone of the acetabulum showed an acetabular index (AC) angle of 27 degrees preoperatively and 14 degrees postoperatively. Statistical analysis proved a significant correlation between the normal postoperative values of the acetabulum/femoral-head index of Heyman and Herndon and the absence of degenerative joint changes at follow-up. 73.5% of the hips had not markedly progressed to secondary osteoarthrosis, but 26.5% of joints had deteriorated: 7.4% of them due to perioperative complications and early postoperative trauma. In 8.8% (severe dysplasia) only partial reorientation was possible, which explains the progression of arthrosis, but 10.2% progressed despite sufficient correction.

Acetabulum↗

PubMed1999

Relevance of the drainage along the linea aspera for the reduction of fat embolism during cemented total hip arthroplasty. A prospective, randomized clinical trial.

The aim of this study was to assess the relevance of drainage placed along the linea aspera for the prevention of fat embolism and cardiopulmonary impairment during the insertion of a cemented stem. We studied 40 patients with coxarthrosis randomly allocated to total hip arthroplasty with proximal drainage or without it. The venting hole for the drainage of the medullary cavity was placed posteriorly, between the greater and the smaller trochanter, in the prolongation of the linea aspera. The heart was monitored intraoperatively by a echocardiography probe positioned in the patient's oesophagus. During the operation we monitored the hemodynamics and blood gas values. Severe embolic events were observed in 85% of the control group and in 20% of the drainage group (P = 0.01). Embolism occurred during the insertion of the femoral component and continued after reduction of the hip joint. After major embolism, the pulmonary shunt values increased significantly in the control group (+22.7%), but there were no marked changes in the drainage group (+7.1%). The logical therapeutic measure to avoid intravasation of bone marrow, fat, and bone debris during the insertion of the femoral component is to prevent the rise of intraosseous pressure. The drainage of the venous system located along the linea aspera significantly reduces the risk of intraoperative embolism and cardiopulmonary impairment.

Aged↗

PubMed1999

Stability of RNA transcripts in post-mortem psychiatric brains.

RNA isolated from frozen human post-mortem brain tissue was used for analysis of five gene products with a recently developed sensitive and competitive RT-PCR technique. Samples varying in post-mortem intervals up to four days from controls, schizophrenics and alcoholics were analyzed. Evaluation of three housekeeping genes, as well as Trk B and Trk C demonstrated that the levels of mRNA transcripts were stable in brain samples at all time periods (one to four days) examined. This observation demonstrates that this RT-PCR protocol is a sensitive and reliable method to study relative amounts of mRNAs. The overall stability of housekeeping transcripts implicates the value of post-mortem brain samples for differential gene expression studies.

Actins↗

PubMed1999

Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction.

In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ channels have been amplified by reverse transcription-polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro, fragments of the three major transcripts alpha1C, alpha1A, and alpha1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca2+ channels. These data support the correlation between alpha1A and P-type Ca2+ channels (G1) and between alpha1E and R-type channels (G2 and G3). By including specific primer pairs for alpha1E the complimentary DNA fragments of indicative regions of alpha1E isoforms are amplified corresponding to the three most variable regions of alpha1E, the 5'-end, the II/III-loop, and the central part of the 3'-end. Although the complementary DNA fragments of the 5'-end of rat alpha1E yield a uniform reverse transcription-polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat alpha1E complementary DNA. It corresponds to the alpha1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of alpha1E. While fragments of a new rat alpha1E isoform are amplified from the 5'-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3'-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the alpha1E expression from a pattern seen in cerebellum (alpha1Ee) to a pattern identified in other regions of the brain (alpha1E-3) is discussed. These data show that: (i) alpha1E is expressed in rat brain as a structural homologue to the mouse and human alpha1E; and (ii) rat cerebellar granule cells in primary culture express a set of alpha1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca2+ channels genes are identified using degenerate oligonucleotide primer pairs, the two isoforms differentiated by the 129 base pair insertion might correspond to the two R-type channels, G2 and G3, characterized in these neurons. Functional studies including recombinant cells with the different proposed isoforms should provide more evidence for this conclusion.

Amino Acid Sequence↗