Jan Olof G. Karlsson

Cloning and expression of a fish α2-adrenoceptor

1 Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α2‐adrenoceptor pharmacology. We now report the cloning of the α2‐F, a fish skin α2‐receptor from the cuckoo wrasse (Labrus ossifagus). 2 Degenerate oligonucleotides corresponding to conserved regions of the human α2‐adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human α2‐adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3 A clone (pTB17BS) consisting of ∼5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and ∼2 kb of 5′‐untranslated sequence. The deduced amino acid sequence of this protein showed 47–57% identity with the human α2‐adrenoceptors and thus appeared to encode a fish α2‐adrenoceptor. 4 In the 5′‐untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α2‐F might be regulated by cyclic AMP, calcium and/or steroids. 5 The α2‐F was expressed in COS‐7 cells and radioligand binding studies were performed with [3H]‐rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 ± 0.1 nm and a Bmax of 5.7 ± 1.0 pmol mg−1 of protein. 6 Competition curves for the displacement of specific [3H]‐rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine> p‐aminoclonidine > B‐HT 920 > (−)‐noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results show that α2‐F has characteristics of both the human α2‐C10 and α2‐C4 and that it might represent an ancestral α2‐adrenoceptor subtype.

Effects of manganese dipyridoxyl diphosphate, dipyridoxyl diphosphate--, and manganese chloride on cardiac function. An experimental study in the Langendorff perfused rat heart.

RATIONALE AND OBJECTIVES.Manganese dipyridoxyl diphosphate (MnDPDP) is a promising contrast agent for magnetic resonance imaging of the liver. The authors explored the possibility that high concentrations of MnDPDP may cause manganese ion (Mn++)-induced side effects on cardiac function. METHODS.Potential cardiodepression by MnDPDP, DPDP– –, and manganese chloride (MnCl2) (100-3,0000 µmol/ L) was investigated in the isolated rat heart, with left ventricular developed (systolic—end-diastolic) pressure and heart rate as the primary indices of cardiac function. RESULTS.During 5-minute exposures, 10% and 50% decreases in left ventricular developed pressure were observed for MnDPDP, 250 µmol/L and 1580 /µmoI/L; DPDP– –, less than 100 µmol/L and 1000 /µmoI/L; MnCl2, 30 /µmol/L and 250 /µmol/L. Heart rate changes were not observed with MnDPDP. Cardiodepression was reversed within 2 minutes during a 14- minute recovery period for all investigated concentrations of MnDPDP and was less rapid for the highest concentrations of MnCl2. CONCLUSIONS.Manganese dipyridoxyl diphosphate is well tolerated in the rat heart at concentrations as high as 200 to 250 limol/L and is approximately 10 times less cardiotoxic than MnCl2. Cardiodepressive effects of MnDPDP in the present rat heart model, perfused in the absence of blood and proteins, are related primarily to the release of free Mn++ions and in part to the simultaneous release of DPDP¯ ¯.

Myocardial manganese elevation and proton relaxivity enhancement with manganese dipyridoxyl diphosphate. Ex vivo assessments in normally perfused and ischemic guinea pig hearts.

Manganese (Mn) dipyridoxyl diphosphate (MnDPDP) is the active component of a contrast medium for liver MRI. By being metabolized, MnDPDP releases Mn2+, which is taken up and retained in hepatocytes. The study examined whether MnDPDP elevates Mn content and enhances proton relaxivity in normal myocardium, but not in ischemic myocardium with reduced coronary flow and impaired metabolism. Isolated guinea pig hearts were perfused at normal flow or low flow, inducing global subtotal ischemia. Ventricular ATP and Mn contents, T1 and T2 were measured. At normal flow tissue Mn content increased from the control level of 4.1 to 70.4 µmol/100g dry wt with MnDPDP (3000 µM), while low‐flow perfusion with MnDPDP (3000 µM) resulted in a Mn content of 16.6 µmol/100 g dry wt. Prolonged ischemia (35 and 90 min) reduced tissue Mn down to the control level. T1 shortening closely paralleled myocardial Mn elevations during both normal and low‐flow perfusion. The use of a Mn2+‐releasing contrast agent like MnDPDP may be a promising principle in MRI assessments of myocardial function and viability in coronary heart disease by revealing a differential pattern of changes in T1 relative to coronary flow, cell Mn uptake and retention, ion channel function and metabolism. Copyright

Characterization of pigment aggregating α2‐adrenoceptors of fish melanophores by use of different agonists after partial irreversible receptor inactivation

1 The affinity for, and the intrinsic efficacy on, postsynaptic melanosome aggregating α2‐adrenoceptors of fish melanophores was studied for B‐HT 920, clonidine, medetomidine, noradrenaline, phenylephrine and UK‐14,304. Investigations were carried out by evaluating the effects of progressive, irreversible inactivation of the α2‐adrenoceptors by benextramine. 2 The double reciprocal plots of equieffective concentrations of B‐HT 920, clonidine, noradrenaline and phenylephrine were linear, which indicated that these compounds exerted their effects, mainly, through interaction with one receptor site. 3 The affinity for the α2‐adrenoceptor selective agonist B‐HT 920, was found to be about 1000 times higher than the affinity for the α1‐adrenoceptor selective agonist phenylephrine. 4 The corresponding plot of equieffective concentrations of medetomidine was not linear, which may indicate that this imidazole compound exerted its effect through more than one receptor site. However, when phenoxybenzamine was used in place of the more selective benextramine, a linear relationship was obtained.

Cardiac Metal Contents After Infusions of Manganese: An Experimental Evaluation in the Isolated Rat Heart

RATIONALE AND OBJECTIVES Manganese dipyridoxyl diphosphate (MnDPDP), a contrast agent for liver MRI, releases free Mn2+ in a graded manner. The aim of the study was to compare the effects of brief versus prolonged infusions of MnDPDP and manganese chloride (MnCl2) on cardiac function, metabolism, Mn accumulation, and tissue metal content. METHODS Isolated perfused rat hearts received 1-minute or 10-minute infusions of MnDPDP (100 microM, 1000 microM) or of MnCl2 (10 microM, 100 microM). Physiologic indices were measured intermittently, and tissue high-energy phosphate compounds and Ca/Fe/Mg/Mn/Zn contents were measured after a standardized Mn washout. RESULTS One-minute and 10-minute infusions induced, respectively, minor and marked depressions of contractile function and corresponding elevations in myocardial Mn content. MnCl2 was markedly more potent than MnDPDP. Ten-minute infusions of the highest concentration of MnDPDP and MnCl2 lowered tissue Mg and elevated tissue Ca (MnCl2), whereas high-energy phosphates were unaffected. CONCLUSIONS Mn uptake after Mn infusion is strongly related to the duration, concentration, and dose of free Mn ions. Differences in Mn accumulation between MnDPDP and MnCl2 were more pronounced after the 10-minute infusion.

Effects of MnDPDP, DPDP--, and MnCl2 on cardiac energy metabolism and manganese accumulation. An experimental study in the isolated perfused rat heart.

RATIONALE AND OBJECTIVES Recent studies indicate that manganese dipyridoxyl diphosphate (MnDPDP) may function as a slow release agent for manganese ions (Mn++) and that MnDPDP is approximately 10 times less potent than manganese chloride (MnCl2) in depressing cardiac function. The authors examined the possibility that MnDPDP and MnCl2 may influence cardiac metabolism and enzyme release and lead to a tissue accumulation of Mn. METHODS Manganese DPDP, DPDP--, or MnCl2 (1000 microM) was infused in isolated rat hearts, which were freeze-clamped at various time intervals during infusion (5 minutes) and recovery (14-minute washout). Enzyme (lactate dehydrogenase) release, tissue high energy phosphates, Mn contents, and physiologic indices were measured at various time intervals. RESULTS No significant differences were noted for: lactate dehydrogenase in the treated groups; tissue creatine phosphate (CrP) and adenosine triphosphate in MnDPDP, DPDP--, and control groups; and tissue Mn in DPDP-- and control groups. Manganese-chloride and MnDPDP-treated hearts accumulated and retained Mn in an 8:1 ratio. Manganese chloride depressed cardiac function more effectively than MnDPDP. CONCLUSIONS The study has shown that: heart tissue uptake and retention of Mn++ is rapid and effective; MnCl2 is approximately eight times more potent than MnDPDP in promoting these effects; and a rise in tissue Mn content to eight to nine times (MnDPDP) or 60 to 70 times (MnCl2) the normal level does not lead to acute side effects on cardiac energy metabolism, function, and enzyme release. The study indicates that MnDPDP may act like a slow release compound for Mn++ ions.

Calmangafodipir [Ca4Mn(DPDP)5], mangafodipir (MnDPDP) and MnPLED with special reference to their SOD mimetic and therapeutic properties.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) participate in pathological tissue damage. Mitochondrial manganese superoxide dismutase (MnSOD) normally keeps ROS and RNS in check. During development of mangafodipir (MnDPDP) as a magnetic resonance imaging (MRI) contrast agent, it was discovered that MnDPDP and its metabolite manganese pyridoxyl ethyldiamine (MnPLED) possessed SOD mimetic activity. MnDPDP has been tested as a chemotherapy adjunct in cancer patients and as an adjunct to percutaneous coronary intervention in patients with myocardial infarctions, with promising results. Whereas MRI contrast depends on release of Mn(2+), the SOD mimetic activity depends on Mn(2+) that remains bound to DPDP or PLED. Calmangafodipir [Ca4Mn(DPDP)5] is stabilized with respect to Mn(2+) and has superior therapeutic activity. Ca4Mn(DPDP)5 is presently being explored as a chemotherapy adjunct in a clinical multicenter Phase II study in patients with metastatic colorectal cancer.

Comparative studies on nerve- and noradrenaline-induced melanosome aggregation within different species of fish.

1. The aggregation of melanosomes within melanophores of the cuckoo wrasse (Labrus ossifagus; belonging to the family Labridae) has, on pharmacological grounds, been shown to be mediated by postsynaptic alpha 2-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. 2. In the present paper we have investigated some American species belonging to the Labridae, Haemulidae, Embiotocidae, Clinidae and Pleuronectidae. 3. In all instances, except in the case of sargo (Haemulidae), we could demonstrate that melanosome aggregation probably was mediated by postsynaptic alpha 2-adrenoceptors which mediate their effect by inhibiting the adenylate cyclase of the melanophores. 4. Although these receptors apparently, on pharmacological grounds, may be classified as alpha 2-adrenoceptors it was also concluded that there is a phylogenetic divergence among these receptors.

Acetylcholine-induced vasodilation may depend entirely upon NO in the femoral artery of young piglets

To characterize agonist‐induced relaxation in femoral artery rings from young piglets, we compared the effect of a NOS‐inhibitor Nω‐nitro‐L‐arginine (L‐NOARG), an NO‐inactivator oxyhaemoglobin (HbO) and a soluble guanyl cyclase(sGC)‐inhibitor 1H‐[1,2,4]Oxadiazolo‐[4,3,‐α]quinoxalin‐1‐one (ODQ) on acetylcholine(ACh)‐induced relaxation. The involvement of K+ channel activation was studied on relaxations induced by ACh, the two NO donors sodium nitroprusside (SNP) and diethylamine (DEA) NONOate, and the cell membrane permeable guanosine 3′5′ cyclic monophosphate (cGMP) analogue 8‐Br‐cGMP. Full reversal of phenylephrine‐mediated precontraction was induced by ACh (1 nM–1 μM) (pD2 8.2±0.01 and Rmax 98.7±0.3%). L‐NOARG (100 μM) partly inhibited relaxation (pD2 7.4±0.02 and Rmax 49.6±0.8%). The L‐NOARG/indomethacin(IM)‐resistant response displayed characteristics typical for endothelium‐derived hyperpolarizing factor (EDHF), being sensitive to a combination of the K+ channel blockers charybdotoxin (CTX) (0.1 μM) and apamin (0.3 μM). ODQ (10 μM) abolished relaxations induced by ACh and SNP. L‐NOARG/IM‐resistant relaxations to ACh were abolished by HbO (20 μM). Ouabain (1 μM) significantly inhibited ACh‐induced L‐NOARG/IM‐resistant relaxations and relaxations induced by SNP (10 μM) and 8‐Br‐cGMP (0.1 mM). A combination of ouabain and Ba2+ (30 μM) almost abolished L‐NOARG/IM‐resistant ACh‐induced relaxation (Rmax 7.7±2.5% vs 23.4±6.4%, with and without Ba2+, respectively, P<0.05). The present study demonstrates that in femoral artery rings from young piglets, despite an L‐NOARG/IM‐resistant component sensitive to K+ channel blockade with CTX and apamin, ACh‐induced relaxation is abolished by sGC‐inhibition or a combination of L‐NOARG and HbO. These findings suggest that relaxation can be fully explained by the NO/cGMP pathway.

MCH-induced pigment aggregation in teleost melanophores is associated with a cAMP reduction.

It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.