137By using the isolated working heart preparation of frog as bioassay, specific
sequences included in the VS-1 peptide [frog and bovine CgA4–16 and CgA47–66,
and bovine CgA1–40 with (CgA1– 40SS) and without an intact disulfide bridge
(CgA1–40SH)] have been tested both under basal and β-adrenergic stimulated con-
ditions. All the fragments studied were able to modulate the cardiac performance in
frog, emphasizing the high phylogenetic conservation of the CgA1–76 sequence.
The most potent fragments in exerting intrinsic negative inotropy and counteraction
against ISO-mediated positive inotropism were the intact VS-1, the fragment
CgA7–57 and the shorter peptide CgA1–40 with the intact disulfide-bridge loop
(Tota et al. 2003 , 2004 ; Corti et al. 2004 ). CgA4–16 and CgA47–66 sequences of
frog and bovine showed lower potencies than those reported for the human recom-
binant VS-1 and CgA7–57 and for the natural configuration of the bovine CgA1–40,
i.e. CgA1–40SS. Frog CgA4–16 (fCgA4–16) resulted the least effective sequence
(Tota et al. 2003 ). Of note, this peptide shows a higher net negative charge than the
bovine counterpart. The ISO-evoked positive inotropy was counteracted by the
bovine CgA1–40SS but not CgA1–40SH. The N- and C-terminal sequences of both
frog and bovine CgA4–16 and CgA47–66 exerted their anti-adrenergic action at
concentrations higher than those required by the fragment which include disulfide-
bridge (CgA1–40SS) (Tota et al. 2003 ). These data suggest that the disulfide-bridge
region of VS-1 is crucial for the cardio-suppression elicited by VS-1 and CgA7–57,
both under basal or adrenergically stimulated conditions (Table 1 ).
2.2 Mechanisms of Action
On the isolated and perfused heart preparation from both eel and frog, the inhibitory
actions of VS-1 and CgA7–57 depend on calcium and potassium channels, being
abolished by lanthanum (a non-specific Ca2+ channel inhibitor), diltiazem (a specific
L-type Ca2+ channel blocker), tetraethylammonium chloride (an inhibitor of the cal-
cium activated-channels), and glibenclamide (an inhibitor of both the sarcolemmal
and mitochondrial ATP-potassium channels), all used at concentrations that per se
do not affect myocardial mechanical performance (Corti et al. 2004 ; Imbrogno et al.
2004 ). These data are in agreement with Aardal and Helle ( 1992 ) which reported a
Ca2+-dependent vasoinhibitory action of VS in human thoracic arteries, and Brekke
and coworkers (Brekke et al. 2002 ) which observed that the vasodilatatory action
induced by CgA1–40 in the bovine coronary arteries is abolished by K+ channels
blockers.
Moreover, in both eel and frog hearts, the VSs-mediated-negative inotropy was
blocked by pre-treatment with inhibitors of cytoskeleton reorganization, such as
cytochalasin-D (a blocker of actin polymerization), wortmannin (an inhibitor of
PI3-kinase/protein kinase B signal-transduction cascade), butanedione 2-monoxime
(an antagonist of myosin ATPase), and N-(6-aminohexil)-5-chloro-1-
naphthalenesulfonamide (W7) (a calcium-calmodulin antagonist), pointing the
Comparative Aspects of CgA-Derived Peptides in Cardiac Homeostasis