[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Differential Cytotoxicities of N-Methyl-β-Carbolinium Analogues of MPP+ in PC12 Cells: Insights into Potential Neurotoxicants in Parkinson's Disease

N-Methylated β-carbolinium cations that can form in vivo from environmental or endogenous β-carbolines are putative neurotoxic factors in Parkinson's disease. The cytotoxicities of 11 N-methylated β-carbolinium cations and N-methyl-4-phenylpyridinium cation (MPP⁺), the experimental parkinsonian neurotoxicant which the carbolinium cations structurally resemble, were examined using rat pheochromocytoma (PC12) cells cultured in “low energy” N-5 medium; cell death was estimated by released lactate dehydrogenase activity and viable cell protein. Of the eight N-monomethylated β-carbolinium cations utilized, only 2-methyl-harmalinium (harmaline-2- methiodide) was as cytotoxic as MPP⁺. Also, three N²(β), N⁸(indole)-dimethylated β-carbolinium cations displayed cytotoxic effects, with the simplest, 2,9-dimethylnorhar- manium, approaching the effectiveness of MPP⁺ in PC12 cells cultured in N-5 medium. However, when PC12 cells grown in higher energy Dulbecco's modified Eagle's medium were utilized with selected effective cations, it was observed that the cultures were relatively resistant to MPP⁺ and 2,9-dimethylnorharmanium, but remained vulnerable to 2-methylharmalinium. The results are interpreted to mean that different cytotoxic mechanisms exist for the two most potent β-carbolinium cations—namely, a mechanism for the 2,9-dimethyl-β-carbolinium species that, as with MPP⁺, is conditional on mitochondrial ATP depletion, but a different (or additional) mechanism for 2- methylharmalinium that is independent of mitochondrial inhibition. The possible accumulation of these cytotoxic cations in Parkinson's disease is discussed in the context of these findings.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

Synthesis and evaluation of 18F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model

Noninvasive imaging of iodide uptake via the sodium/iodide symporter (NIS) has received great interest for evaluation of thyroid cancer and reporter imaging of NIS-expressing viral therapies. In this study, we investigate ¹⁸F-labeled hexafluorophosphate (HFP or PF₆^?) as a high-affinity iodide analog for NIS imaging. ¹⁸F-HFP was synthesized by radiofluorination of phosphorus pentafluoride·N-methylpyrrolidine complex and evaluated in human NIS (hNIS)-expressing C6 glioma cells and a C6 glioma xenograft mouse model. ¹⁸F-HFP was obtained in radiochemical yield of 10?±?5%, radiochemical purity of >96% and specific radioactivity of 604?±?18?MBq/µmol. Specific uptake of ¹⁸F-HFP and high affinity of ¹⁹F-HFP were observed in hNIS+ C6-glioma cells. PET imaging showed robust uptake of ¹⁸F-HFP in NIS-expressing tissues (thyroid, stomach, and hNIS+ C6 glioma xenografts), and the uptake of ¹⁸F-HFP was blocked by NaClO₄ pretreatment. Specific accumulation in hNIS-expressing xenograft (hNIS+) was observed relative to isogenic control tumor (hNIS?). Clearance of ¹⁸F-HFP was predominantly through renal excretion. The biodistribution showed consistent results with PET imaging. Minimal bone uptake was observed over 2?h period post-injection, indicating excellent in vivo stability of ¹⁸F-HFP. Although improvement in specific radioactivity is desirable, the results indicate that ¹⁸F-HFP is a promising candidate radiotracer for further evaluation for NIS imaging.     

Synthesis and structures of aryloxo- and binaphthoxogermanium(IV) alkyl iodide complexes

The germanium(II) aryloxide complexes (S)-[Ge{O₂C₂₀H₁₀-(SiMe₂Ph)₂-3,3′}{NH₃}] (1) and [Ge(OC₆H₃Ph₂-2,6)₂] (2) react with either Bu^tI or MeI to yield the corresponding germanium(IV) compounds (S)-[Ge{O₂C₂₀H₁₀-(SiMe₂Ph)₂-3,3′}{Bu^t}{I}] (3), (S)-[Ge{O₂C₂₀H₁₀-(SiMe₂Ph)₂-3,3′}{Me}{I}] (4), [Ge(OC₆H₃Ph₂-2,6)₂(Bu^t)(I)] (5), and [Ge(OC₆H₃Ph₂-2,6)₂(Me)(I)] (6). Compound 6 reacts with 2,6-diphenylphenol to yield [Ge(OC₆H₃Ph₂-2,6)₃(Me)] (7), while 3-5 do not. The X-ray crystal structures of 3-5 and 7 were determined, and 3-5 represent the first structurally characterized germanium(IV) species having germanium bound to both oxygen and iodine.     

Iodate and iodide effects on iodine uptake and partitioning in rice (Oryza sativa L.) grown in solution culture

In the Xinjiang province of western China, conventional methods of iodine (I) supplementation (i.e, goiter pills and iodinated salt) used to mitigate I deficiencies were ineffectual. However, the recent addition of KIO₃ to irrigation waters has proven effective. This study was conducted to determine the effects of I form and concentration on rice (Oryza sativa L.) growth, I partitioning within the plant, and ultimately to assist in establishing guidelines for incorporating I into the human food chain. We compared IO₃ ⁻ vs. I⁻ in order to determine how these chemical species differ in their biological effects. Rice was grown in 48 L aerated tubs containing nutrient solution and IO₃ ⁻ or I⁻ at 0, 1, 10, or 100 μM concentrations (approximately 0, 0.1, 1, and 10 mg kg⁻¹ I). The IO₃ ⁻ at 1 and 10 μM had no effect on biomass yields, and the 100 μM treatment had a small negative effect. The I⁻ at 10 and 100 μM was detrimental to biomass yields. The IO₃ ⁻ treatments had more I partitioning to the roots (56%) on average than did the I⁻ treatments (36%), suggesting differences in uptake or translocation between I forms. The data support the theory that IO₃ ⁻ is electrochemically or biologically reduced to I⁻ prior to plant uptake. None of the treatments provided sufficient I in the seed to meet human dietary requirements. The I concentration found in straw at 100 μM IO₃ ⁻ was several times greater than seed, and could provide an indirect source of dietary I via livestock feeding on the straw. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sonochemical synthesis of nano-sized metal-organic lead(II) polymer: A precursor for the preparation of nano-structured lead(II) iodide and lead(II) oxide

Nanoparticles of a new Pb^II metal-organic polymer, [Pb(μ-pyr)(μ-I)₂]_n (1), with a net-like morphology have been synthesized by the reaction of pyrazine with Pb(NO₃)₂ and NaI via sonochemical irradiation. Nano-structured PbI₂ and PbO were synthesized from compound 1 by calcination at argon and air atmospheres, respectively. The structure of 1 was determined by single crystal X-ray crystallography and the nano-structures were characterized by X-ray powder diffraction (XRD) and scanning electron microscopy (SEM). The thermal stability of nano-sized and single crystalline samples of 1 were studied and compared.     

Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.     

Different tissue responses for iodine and iodide in rat thyroid and mammary glands

This research describes the effects of short-term elemental iodine (I₂) and iodide (I⁻) replacement on thyroid glands and mammary glands of iodine-deficient (ID) Sprague-Dawley female rats. Iodine deficiency causes atypical tissue and physiologic changes in both glands. Tissue histopathology and the endocrine metabolic parameters, such as serum TT₄, tissue and body weights, and vaginal smears, are compared. A moderate reduction in thyroid size from the ID control (IDC) was noted with both I⁻ and I₂, whereas serum total thyroxine approached the normal control with both I⁻ and I₂, but was lower in IDC. Thyroid gland IDC hyperplasia was reduced modestly with I₂, but eliminated with I⁻. Lobular hyperplasia of the mammary glands decreased with I₂ and increased with I⁻ when compared with the IDC; extraductal secretions remained the same as IDC with I₂, but increased with I⁻; and periductal fibrosis was markedly reduced with I₂, but remained severe with I⁻. Thus, orally administered I₂ or I⁻ in trace doses with similar iodine availability caused different histopathological and endocrine patterns in thyroid and mammary glands of ID rats. The significance of this is that replacement therapy with various forms of iodine are tissue-specific.     

Modulation of MPP+ uptake by procyanidins in Caco-2 cells: involvement of oxidation/reduction reactions

It is becoming increasingly evident that the absorption of certain nutrients and drugs and their effects are largely influenced by the concomitant ingestion of other substances. As various xeno- and endobiotics belong to the class of organic cations, the aim of this work was to study the modulation of the intestinal apical uptake of organic cations by diet procyanidins.Five procyanidin fractions with different structural complexity were obtained after fractionation of a grape seed extract. The effect of these compounds on 1-methyl-4-phenylpyridinium (MPP⁺) uptake was evaluated in Caco-2 cells.Apical uptake of ³H-MPP⁺ by Caco-2 cells was increased by a 60 min exposure to 600 μg ml⁻¹ of procyanidin fractions, that increase being positively related with procyanidins structural complexity. It was verified that ³H-MPP⁺ uptake increased with preincubation time. It was speculated that procyanidins were oxidized during preincubation, this change could interfered with transport activity. Tested oxidizing agents showed that the redox state of the transporter could affect its activity. Additionally, trans-stimulation experiments showed that catechin and fraction I (the simpler fraction) can use the same transporter as MPP⁺. The results are compatible with the hypothesis of these compounds being competitive inhibitors of MPP⁺ transport.In conclusion, procyanidins are capable to modulate MPP⁺ apical uptake in Caco-2 cells, this transport being most probably modulated through oxidation-reduction phenomena. Interactions between these compounds and drugs present in the diet may affect their absorption and bioavailability. Both the concentration and complexity of the procyanidin compounds should be taken into account in medical practice.     

Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (Lmna^HG/+). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (Lmna^nHG/+) develop progeria-like disease phenotypes. The fact that Lmna^nHG/+ mice manifest disease raised the possibility that the beneficial effects of an FTI in Lmna^HG/+ mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both Lmna^HG/+ and Lmna^nHG/+ mice. In Lmna^HG/+ mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in Lmna^nHG/+ mice. The failure of the FTI to ameliorate disease in Lmna^nHG/+ mice supports the idea that the beneficial effects of an FTI in Lmna^HG/+ mice are due to the effect of drug on the farnesylation of progerin.     

Dihydropyridine Action on Voltage-dependent Potassium Channels Expressed in Xenopus Oocytes

Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca²⁺ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K⁺ channels. We examined the effects of DHPs on the Shaker K⁺ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K⁺ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.     

Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae

The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na⁺ and K⁺ was demonstrated, with maximum stimulation at 20–40 mM concentrations. The divalent cations Mg²⁺ and Ca²⁺ stimulated proteolytic activity at low concentrations (between 0 and 10 mM) but inhibited activity at higher concentrations. The divalent cations Zn²⁺, Cu²⁺, Fe²⁺, and Co²⁺ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.     

Growth history and ecology of the Atlantic surf clam,Spisula solidissima (Dillwyn), as revealed by stable isotopes and annual shell increments

The shells of the Atlantic surf clam, Spisula solidissima (Dillwyn), contain a record of both life history and environmental changes. These shell records were investigated using oxygen and carbon stable isotopic analyses (δ¹⁸O, δ¹³C) and shell growth increment analyses. δ¹⁸O variations across annual shell increments reflect the yearly cycle of sea-water temperatures off the New Jersey coast, further documenting the proposed annual periodicity of the major shell increments. The 11-yr shell record analyzed here confirms that shell growth is most rapid in spring-early summer, slow in late summer-fall, and extremely slow or non-existent in winter. Shell growth appears to occur in isotopic equilibrium with sea water and measured δ¹⁸O values are used to refine the aragonite-water temperature scale. Variations in the timing of annual growth increment formation are noted as well as ontogenetic effects upon the range of isotopic values recorded in shell carbonate. Both the δ¹⁸O and δ¹³C profiles are influenced by changes in the sea-water temperature regime over the 11-yr period studied (1965–1976) and record these effects in the shell. The combination of stable isotope and growth increment analyses provides a powerful tool for interpreting the shell records of both modern and fossil molluscs.     

An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min^-1 M^-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (K_p) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K_d value to 571 mM from the native enzyme (K_d = 150 mM). Although arginine-modified enzyme reacts with H₂O₂ to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min^-1 M^-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H₂O₂ suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.     

Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure–activity analyses: dissociation of antihypertensive and bradycardic effects

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr¹-Asn²-Trp³-Pro⁴-His⁵-Pro⁶-Gln⁷-Ile⁸-Pro⁹-Pro¹⁰). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala³, Bj-PRO-10c Ala⁷, Bj-PRO-10c Ala⁸ were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala², Bj-PRO-10c Ala⁴, Bj-PRO-10c Ala⁵, Bj-PRO-10c Ala⁹, and Bj-PRO-10c Ala¹⁰ kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala¹ and Bj-PRO-10c Ala⁶ were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr¹ and Pro⁶ residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro⁶ → Ala⁶ mutant is probably due to the fact that in the parent peptide the His⁵-Pro⁶ bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.