Stefan Quasthoff

Calcium potentials and tetrodotoxin-resistant sodium potentials in unmyelinated C fibres of biopsied human sural nerve

Compound action potentials and electrotonic responses to 150 ms current pulses were recorded from isolated nerve fascicles of human sural nerve biopsies. Compound action potentials in normal bathing solution were characterized by previously described A beta, A delta and C fibre components. In addition, tetrodotoxin-resistant sodium- or calcium-dependent potential components were found when a mixture of tetrodotoxin and the potassium channel blockers 4-aminopyridine and tetraethylammonium was added to the bathing solution. In contrast to tetrodotoxin-sensitive action potentials, tetrodotoxin-resistant sodium- or calcium-dependent potentials could be recorded in the presence of high extracellular potassium concentrations (10-20 mM). Calcium action potentials were found to be sensitive to specific pharmacological antagonists or agonists of L-, N- and P-type calcium channels. Lidocaine, cadmium, verapamil and capsaicin showed unspecific blocking effects on calcium and tetrodotoxin-resistant potentials. Tetrodotoxin-resistant action potentials seem to originate from unmyelinated C fibres since a clear correlation was found between the number of C fibres and the amplitude of tetrodotoxin-resistant calcium and sodium spikes in preparations with different axon type composition. The evidence for tetrodotoxin-resistant Na+ and Ca2+ spikes in peripheral human axons offers new possibilities for a better understanding and/or treatment of abnormalities in the excitability of damaged or diseased peripheral nerves.

X-linked dominant Charcot-Marie-Tooth neuropathy: clinical, electrophysiological, and morphological phenotype in four families with different connexin32 mutations(1).

The sensorimotor neuropathy of the Charcot-Marie-Tooth type (CMT) is the most common hereditary disorder of the peripheral nervous system. The X-linked dominant form of CMT (CMTX) is associated with mutations in the gene for the gap junction protein connexin32. We examined four CMTX pedigrees two of which had potentially novel mutations in the only coding exon of connexin32. One previously unreported missense mutation, Ala39Val, was found in a family displaying a CMT phenotype with additional upper limb postural tremor reminiscent of a Roussy-Lévy syndrome. A novel single base insertion, 679insT, is among the first mutations found in the fourth transmembrane domain of connexin32. Frameshift and premature stop of translation are supposed to result in a non-functional carboxy-terminus. Two further families had the known missense mutations Arg15Trp and Arg22Gln. Several female carriers were found normal on clinical presentation, however, the genotype was paralleled by decreased nerve conduction velocities (NCV) and slowed central conduction of brain stem auditory evoked responses (BAER). Median motor NCVs showed mild (in women) to intermediate (in males) reduction, indicating a peripheral neuropathy with a predominating axonal component. Nerve biopsy findings were consistent with the electrophysiological data showing a marked loss of large myelinated fibres and clusters of regenerating axons. Electron microscopy revealed various alterations of the axoglial attachment zone. This suggests defective axon-Schwann cell interactions which may induce the axonopathy in CMTX.

Differences in the sensitivity to purinergic stimulation of myelinating and non‐myelinating Shwann cells in peripheral human and rat nerve

Schwann cells of the peripheral nervous system are distinguished by morphological and functional criteria in myelinating and non‐myelinating subtypes. We and others have previously reported that Schwann cells in isolated peripheral human and rat nerve respond to extracellular application of ATP with a rise in the intracellular free calcium concentration [Ca2+]i. In the present study, the receptors mediating these Ca2+ transients have been investigated in myelinating and non‐myelinating Schwann cells of intact fascicles of isolated human sural nerves, rat ventral roots, and rat vagus nerves. Microfluorometry and confocal laser scanning was used on preparations stained with the Ca2+‐sensitive dyes Calcium Green‐1 and Fura Red. In myelinating Schwann cells of human and rat nerves, the ATP‐induced rise of [Ca2+]i resulted from the activation of a P2Y2 purinoceptor subtype (rank order of potency: UTP ≥ ATP ≫ 2‐MeSATP = ADP). In contrast, in non‐myelinating Schwann cells, Ca2+ transients were produced by activation of a P2Y1 purinoceptor subtype (rank order of potency: 2‐MeSATP > ATP > ADP ≫ UTP). The P1 agonist adenosine and α,β‐meATP did not evoke Ca2+ signals. Ca2+ transients in both types of Schwann cells were found to be due to Ca2+ release from cyclopiazonic acid‐sensitive intracellular stores. However, inhibition by suramin was only found in non‐myelinating Schwann cells. These findings indicate that mammalian Schwann cells express phenotype‐specific P2Y receptor subtypes. GLIA 23:374–382, 1998.

X-linked dominant Charcot-Marie-Tooth disease: nerve biopsies allow morphological evaluation and detection of connexin32 mutations (Arg15Trp, Arg22Gln).

Abstract X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the connexin32 gene on Xq13. Because of overlapping morphological and clinical data, CMTX patients often meet the criteria of autosomal-dominant CMT2, the neuronal type of CMT. Hence, it might be useful to analyse the connexin32 gene in suspected CMT2 patients when there is no male-to-male transmission. We selected a cohort of 30 patients who were considered having CMT2 on the basis of previous clinical and histopathological evaluation. DNA was extracted from paraffin-embedded sural nerve biopsy samples and screened for connexin32 mutations to verify the possible diagnosis of CMTX. In 2 patients mutations were found corresponding to amino acid substitutions of arginine for tryptophan in codon 15 and arginine for glutamine in codon 22 of connexin32. This study illustrates that archival material allows genetic classification of suspected CMT cases. Furthermore, there is additional proof that connexin32 mutations represent the underlying genetic defect in some cases of predominantly neuronal CMT.

Expression of Messenger RNA of the Cardiac Isoforms of Troponin T and I in Myopathic Skeletal Muscle

In the absence of clinical signs, elevated values of the cardiac isoforms of troponin T (cTnT) and I (cTnI) can be found in the serum samples of some patients with skeletal muscle myopathies; the cause is unclear. We studied the messenger RNA (mRNA) expression of cTnT and cTnI in the skeletal muscles of 24 patients with histologically proven myopathies and in 18 patients in whom a myopathy could be excluded. For cTnT- and cTnI-mRNA determination, we designed specific primer pairs for nested polymerase chain reaction. After amplification, the products were digested with 2 restriction enzymes and visualized. We found cTnT mRNA in 7 skeletal muscle biopsy specimens (6 patients with Duchenne muscular dystrophy, 1 patient with a primary sarcoglycanopathy) and cTnI mRNA in 6 (5 with Duchenne muscular dystrophy, 1 patient with a histologically negative biopsy). The mRNA of the cardiac isoforms, cTnT and cTnI, is expressed in the skeletal muscles of patients with Duchenne muscular dystrophy, but also in some other myopathies. Further studies are needed to show whether the mRNA is translated into the protein, but serum levels of cTnT and cTnI in patients with Duchenne muscular dystrophy would seem to indicate this.

The role of axonal ion conductances in diabetic neuropathy: A review

Diabetic neuropathy is a common complication in diabetes mellitus. Diabetic neuropathy is accompanied by alterations in axonal excitability, which can lead to either “positive” (paresthesia, dysesthesia, pain) and/or “negative” (hypesthesia, anesthesia) symptoms. The mechanisms underlying these alterations in axonal excitability are not well understood. Clinical tests reveal reduced nerve conduction velocity and axonal loss, but fail to explain nerve excitability. Many different factors have been suggested in relation to the pathophysiology of diabetic neuropathy. There are probably as many factors as there are different clinical pictures in diabetic neuropathy. Nevertheless, it seems that hyperglycemic hypoxia is mainly responsible for the electrophysiological changes seen in damaged diabetic nerves. This article summarizes experimental data indicating that a dysfunction of ion conductances, especially voltage‐gated ion channels, could contribute to abnormalities in the generation and/or conduction of action potentials in diabetic neuropathy.

Hyperglycaemic hypoxia alters after-potential and fast K+ conductance of rat axons by cytoplasmic acidification.

1. The effects of hyperglycaemic hypoxia (a condition possibly involved in the pathogenesis of diabetic neuropathy) on the depolarizing after‐potential and the potassium conductance of myelinated rat spinal root axons were investigated using electrophysiological recordings from intact spinal roots and from excised, inside‐out axonal membrane patches. 2. Isolated spinal roots were exposed to hypoxia in solutions containing normal or high glucose concentrations. The depolarizing after‐potential of compound action potentials was only enhanced in spinal roots exposed to hyperglycaemic (25 mM D‐glucose) hypoxia. A maximal effect was seen in bathing solutions with low buffering power. 3. The depolarizing after‐potential was also enhanced by cytoplasmic acidification after replacement of 10‐30 mM chloride in the bathing solution by propionate. 4. Multi‐channel current recordings from excised, inside‐out axonal membrane patches were used to study the effects of cytoplasmic acidification on voltage‐dependent K+ conductances with fast (F channels) and intermediate (I channels) kinetics of deactivation. 5. F channels were blocked by small changes in cytoplasmic pH (50% inhibition at pH 6.9). I channels were much less sensitive to intra‐axonal acidification. 6. In conclusion, our data show that hyperglycaemic hypoxia enhances the depolarizing after‐potential in peripheral rat axons. The underlying mechanism seems to be an inhibition of a fast, voltage‐dependent axonal K+ conductance by cytoplasmic acidification. This alteration in membrane conductance may contribute to positive symptoms in diabetic neuropathy.

K+ channel openers suppress myotonic activity of human in vitro

Abstract Isolated fibre bundles from myotonic human skeletal muscle showed after-contractions and spontaneous mechanical activity. The K + channel openers cromakalim (10–100 μmol/1) and EMD 52962 (1–10 μmol/1) completely suppressed these abnormalities in mechanical activity. Voltage-clamp experiments revealed that cromakalim (100 μmol/1) increased the membrane K + conductance of isolated, non-myotonic human skeletal muscle fibres 4-fold; Cl − conductance was not altered. The data show that myotonia is suppressed by an increase in membrane K + conductance.

Clinical phase II evaluation of paclitaxel in combination with cisplatin in metastatic or recurrent squamous cell carcinoma of the head and neck

BACKGROUND Paclitaxel as single agent has shown marked activity in several malignancies. The aim of the present phase II trial was to determine the activity of paclitaxel/cisplatin in patients with metastatic or recurrent squamous cell carcinoma of the head and neck. PATIENTS AND METHODS 200 mg/m2 paclitaxel was administered over three hours followed by cisplatin (100 mg/m2), repeated every 22 days. Twenty-eight patients were entered and received a total of 99 cycles (median 2, range 1-6). All patients were evaluable for toxicity, and 25 for response. RESULTS Hematologic toxicities included leukopenia CTC grade 3 in 13 patients, and grade 4 in five patients, neutropenia grade 3 in nine patients, and grade 4 in eight patients, grade 3 anemia and grade 2 thrombocytopenia in one patient each. Non-hematologic toxicities included hypotension grade 2 (six patients), grade 3 (four patients), and grade 4 (two patients). A decline in renal function was observed in 15 courses and 10 patients, leading to a median delay of 2.5 days. Neurosensory and neuromotor toxicity grade 1 were observed in 13 patients (grade 2: 12 patients; grade 3: one patient), myalgia grade 3 in one patient, asthenia grade 3 in two and grade 4 in one patient. Partial responses were observed in 12 patients for an overall response rate of 48% (95% CI: 28%-68%) with a median response duration of 6.5 months (range 1-10 months). Stable disease was observed in seven patients, of who two also had clinical benefit. CONCLUSIONS Paclitaxel 200 mg/m2 administered over three hours combined with cisplatin 100 mg/m2 is an active regimen warranting further evaluation.

Paclitaxel Administered over 3 h Followed by Cisplatin in Patients with Advanced Head and Neck Squamous Cell Carcinoma: A Clinical Phase I Study

We have performed a clinical phase I trial of a combination treatment with paclitaxel given as 3-hour infusion and cisplatin to determine the maximum tolerated dose and the dose-limiting toxicity in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Treatment was repeated every 21 days. Doses administered ranged from 135 mg/m2 paclitaxel/75 mg/m2 cisplatin to 250 mg/m2 paclitaxel/100 mg/m2 cisplatin. Twenty-four patients have been entered into this study. The maximum tolerated dose was determined to be 225-250 mg/m2 paclitaxel/100 mg/m2 cisplatin. The dose-limiting toxicity of this regimen was myelosuppression (granulocytopenia). Neurosensory and neuromotor toxicity was moderate. However, analyses of threshold electrotonus studies indicated subclinical neurotoxicity in most patients. One patient receiving 200 mg/m2 paclitaxel/100 mg/m2 cisplatin developed grade 3 motor-neurotoxicity. Orthostatic hypotension was observed in 8 patients receiving doses of 200 mg/m2 paclitaxel/100 mg/m2 cisplatin or higher. Objective responses were observed at paclitaxel 175 mg/m2/ cisplatin 100 mg/m2 (n = 5; complete response in 1 patient), paclitaxel 200 mg/ m2/cisplatin 100 mg/m2 (n = 3; partial response in 3 patients) and at paclitaxel 225 mg/m2/cisplatin 100 mg/m2 (n = 8; partial response in 1 patient). Eleven additional patients had stable disease. We conclude that paclitaxel administered as a 3-hour infusion followed by cisplatin is an active regimen in advanced head and neck cancer and that orthostatic hypotension may be a potentially significant clinical toxicity.